Nitric Oxide Mediated Signal Transduction in Networks of Human ...

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01.06.2010 Hartinger

Nitric Oxide Mediated Signal Transduction in Networks of
Human Model Neurons
Thesis
Submitted in partial fulfillment of the requirements for the degree
Doctor of Philosophy (PhD)
Division of Cell Biology, Institute of Physiology
University of Veterinary Medicine Hannover
and
Center for Systems Neuroscience Hannover
Awarded by University of Veterinary Medicine Hannover
by
Million Adane Tegenge
born in
Nekemte / Ethiopia
Hannover, Germany, 2010

Supervisor: Prof. Dr. Gerd Bicker
Division of Cell Biology, Institute of Physiology,
University of Veterinary Medicine Hannover
Advisory group: Prof. Dr. Herbert Hildebrandt
Department of Cellular Chemistry
Hannover Medical School
Prof. Dr. Stephan Steinlechner
Department of Zoology
University of Veterinary Medicine Hannover
First evaluation: Prof. Dr. Gerd Bicker
Prof. Dr. Herbert Hildebrandt
Prof. Dr. Stephan Steinlechner
`
Second evaluation: PD Dr. Wolfgang Blenau
Institute of Biochemisty and Biology
University of Potsdam, Germany
Date of final examination: April 09, 2010
The present work was supported by grants from the DFG (FG 1103; BI 262/16-1) and BMBF
(0313732; 013925D) to G. Bicker. M.A. Tegenge received a Georg-Christoph-Lichtenberg
scholarship from the Ministry for Science and Culture of Lower Saxony.

Dedicated to Lydia and Betania

Publications:
Tegenge, M. A., and Bicker, G. (2009). Nitric oxide and cGMP signal transduction positively
regulates the motility of human neuronal precursor (NT2) cells. J. Neurochem 110, 1828-1841.
Tegenge, M. A., Stern, M., and Bicker, G. (2009). Nitric oxide and cyclic nucleotide signal
transduction modulates synaptic vesicle turnover in human model neurons. J. Neurochem. 111,
1434-1446.
Tegenge, M.A., Rockel, T.D., Fritsche, E. and Bicker G. Nitric oxide signaling as regulator of
human neuronal progenitor cell migration (In preparation).
Podrygajlo, G., Tegenge, M. A., Gierse, A., Paquet-Durand, F., Tan, S., Bicker, G., and Stern, M.
(2009). Cellular phenotypes of human model neurons (NT2) after differentiation in aggregate
culture. Cell Tissue Res 336, 439-452.
Abstract presented in scientific meetings:
Tegenge, M.A and Bicker G. (2009). Nitric oxide and cyclic guanosine-monophosphate signal
transduction facilitates cell motility and neurite outgrowth in differentiating human model neurons,
Meeting of the Society for Neuroscience, Chicago, USA.
Tegenge, M.A and Bicker G. (2009). Pre-synaptic Vesicle Exocytosis in Human Model Neurons
Generated by Spherical Aggregate Culture Method, 8 th Meeting of the German Neuroscience
Society, Gottingen, Germany.
Tegenge, M.A. and Bicker G. (2008). Nitric oxide signal transduction facilitates the migration of
human neuronal precursor (NT2) cells, FENS Abstr. vol.4, 180.3, 6 th FENS Forum of European
Neuroscience, Geneva, Switzerland.
Tegenge, M.A. and Bicker G. (2008). Nitric oxide signal transduction facilitates the migration of
human neuronal precursor (NT2) cells, Brainstorming IV, Center for Systems Neuroscience
Hannover, Germany.

Table of content
1. Introduction 1
1.1. Nitric oxide signal transduction 1
1.1.1. Discovery of NO 1
1.1.2. Synthesis of NO and activation of downstream effectors 1
1.2. NO and early stages of CNS development 3
1.2.1. Neuronal precursor proliferation 3
1.2.2. Neuronal migration 4
1.2.3. Neuronal differentiation 5
1.2.4. Synaptogenesis 6
1.3. NO and adult neurogenesis 7
1.4. NO signaling in neurodegenerative diseases 8
1.5. Human neuronal stem cells as a model of developing nerve cells 9
1.6. Hypothesis and aim of the study 11
2. Discussion 12
2.1. Nitric oxide and cGMP signal transduction positively regulates
the motility of human neuronal precursor (NT2) cells 12
2.2. Nitric oxide and cyclic nucleotide signal transduction modulates
synaptic vesicle turnover in human model neurons 13
2.3. Nitric oxide signaling as regulator of human neuronal progenitor cell migration 16
References 18
Acknowledgments 30
Summary 31
Zusammenfassung 33
3. Publications
3.1. Nitric oxide and cGMP signal transduction positively regulates the motility of human
neuronal precursor (NT2) cells, Tegenge M.A. and Bicker G. (2009), J. Neurochem. 110, 1828-1841.
3.2. Nitric oxide and cyclic nucleotide signal transduction modulates synaptic vesicle turnover in
human model neurons, Tegenge M.A. et al. (2009), J. Neurochem. 111, 1434-1446.
3.3. Nitric oxide signaling as regulator of human neuronal progenitor cell migration,
Tegenge M.A. et al. (In preparation).

List of abbreviations
AD, Alzheimer’s disease
BrdU, 5-bromo-2’-deoxyuridine
cADPR, cyclic adenosine diphosphate ribose
cGMP, cylic guanosine-monophosphate
CICR, Ca (2+)-induced Ca (2+) release
CNS, central nervous system
CNGs, cyclic nucleotide gated ion channels
CREB, cyclic adenosine monophosphate response element binding protein
DG, dentate gyrus
DRG, dorsal root ganglion
EC, embryonal carcinoma cells
EDRF, endothelial derived relaxing factor
eNOS, endothelial nitric oxide synthase
ES, embryonic stem cells
FM1-43, N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide
GTP, guanosine triphosphate
HDAC, histone deacetylase
hNPCs, human neural progenitor cells
iNOS, inducible nitric oxide synthase
IR, immunoreactive
L-NAME, nitro-L-arginine methyl ester
MAP, microtubule-associated protein
MPTP, 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine
7NI, 7-nitroindazole
NMDA, N-methyl D-aspartate
NO, nitric oxide
NOC-18, (2, 2-(Hydroxynitrosohydrazino) bis-ethanamine])
NOS, nitric oxide synthase
nNOS, neuronal nitric oxide synthase
NSC, neuronal stem cells
6-OHDA, 6-hydroxydopamine
PBS, phosphate-buffered saline
PDEs, cyclic nucleotide phosphodiesterase

PKA, protein kinase A
PKG, protein kinase G
PSD, postsynaptic density
RA, retinoic acid
RyRs, ryanodine receptor
sGC, soluble guanylyl cyclase
SVZ, subventricular zone

1. Introduction
1.1. Nitric oxide signal transduction pathway
1.1.1. Discovery of NO
Studies in 1970s have demonstrated that the smooth muscle-relaxing effects of nitroglycerine and
other organic nitrate vasodilators involve an active metabolite, nitric oxide (NO), whose properties
are very smilar to a substance named endothelial derived relaxing factor (EDRF) (Arnold et al.,
1977; Katsuki et al., 1977). Furchgott and Zawadzki (1980) elegantly showed that relaxation of
blood vessels by acetylcholine is abolished when the endothelial layer is removed, but can be
rescued by reapplying endothelial cells to the smooth muscle layer. These studies indicated that a
diffusible molecule from the endothelium mediates muscle and blood vessels relaxation, and
attempt to isolate this molecule was proved to be difficult. Subsequent studies directly confirmed
that chemically detectable levels of NO released from endothelial cells account for all EDRF
activity (Murad, 1986; Ignarro, 1987; Palmer, 1987). In 1998, a group of scientists that include
Robert Furchgott, Louis Ignarro, and Ferid Murad have been awarded the Nobel Prize in Medicine
or Physiology for their discoveries concerning NO as a signaling molecule in the cardiovascular
system. This was the first discovery that revealed a simple gas can act as a signaling molecule in an
animal, which initiated a remarkable number of researches in various field.
In the brain Garthwaite et al. (1988) demonstrated that EDRF like diffusible messenger increased
the level of cGMP in dissociated culture of neonatal cerebellar cells. The formation of NO from
arginine was directly demonstrated in cerebellar slices (Bredt and Synder, 1989). Subsequent
studies established NO as unconventional retrograde neurotransmitter that has been implicated to
modulate synaptic plasticity, memory formation, cell proliferation, migration, differentiation and
synaptogenesis (reviewed by Müller, 1997; Enikolopov et al., 1999; Bicker, 2001; 2005; Boehning
and Snyder, 2003; Cárdenas et al., 2005; Garthwaite, 2008). However, only limited data are
available addressing the role of NO signal transduction during the development of the central
nervous system (CNS) in both human and experimental animals.
1.1.2. Synthesis of NO and activation of downstream effectors
NO is synthesized by nitric oxide synthase (NOS), an enzyme that converts L-arginine into
equimolar quantities of NO and L-citrulline in the presence of several cofactors (Figure 1). There
are three isoforms of NOS; neuronal NOS (nNOS or NOS I), endothelial NOS (eNOS or NOS III)
and inducible NOS (iNOS or NOS II). Two of these enzymes are constitutively expressed in
1

neuronal and endothelial cells; therefore known as neuronal and endothelial NOS, respectively
(Knowles and Moncada, 1994, Förstermann et al., 1995).
The eNOS is found in the caveoli of endothelial cells and is activated following cholinergic
stimulation and the consequent increase of intracellular calcium. The nNOS is found in the
cerebellum, the cerebral cortex, spinal cord and in various ganglion cells of the autonomic nervous
system (Bredt et al., 1990; Grzybicki et al., 1996; Zhou and Zhu, 2009). The nNOS is physically
associated with the N-methyl D-aspartate (NMDA) receptor and postsynaptic density protein-95
(PSD-95) which suggests NMDA activation as a precondition for the synthesis of NO (Garthwaite
et al., 1988, Garthwaite, 2008). The inducible NOS (iNOS or NOS II) is formed mainly in immune
cells, such as macrophages and glial cells (Agullo and Garcia, 1992; Simmons and Murphy, 1992).
Unlike eNOS and nNOS, synthesis of iNOS mRNA is induced by lipopolysaccharide that activate
its receptors on the surface of macrophages and astrocytes (Baltrons et al., 2003, Rettori et al.,
2009).
Although it is difficult to accurately determine the exact physiological concentrations of NO, recent
studies suggested that it may range from 100 pM to 5 nM, orders of magnitude lower than
previously thought (Hall and Garthwaite, 2009). Hence, activation of its downstream targets
depends on local concentration of NO and availability of target molecules (Madhusoodanan and
Murad, 2007). The major low concentration physiological target enzyme for NO is the enzyme,
soluble guanylyl cyclase (sGC) (Garthwaite, 2008). sGC is a heterodimeric protein composed of α
and β subunits. There are two forms of α subunits; the major occurring α1 and the less abundant α2
which are dimerized to common β subunit. The αβ-heterodimer comprises a haem-binding region
and a catalytic domain (Haghikia et al., 2007, Garthwaite, 2008). Binding of NO to the heme
domain leads to the conversion of guanosine triphosphate (GTP) to cylic guanosine-monophosphate
(cGMP) (Figure 1). Genomic deletion of the β1 subunit of sGC has been implicated to completely
disrupt the NO-cGMP signaling whereas mice lacking both α and β subunits has been employed to
dissect cGMP-independent action of NO (Friebe and Koesling, 2009). Potential target proteins
downstream of cGMP includes protein kinase G (PKG), cyclic nucleotide gated ion channels
(CNGs) and cyclic nucleotide phosphodiesterase (PDEs). Each of these downstream effectors then
transmit the signals to an array of intracellular signaling molecules, thereby regulating
neurotransmission, proliferation, cell migration, differentiation, axon outgrowth and guidance
(Madhusoodanan and Murad , 2007).
In pathological conditions (such as brain ischaemia or neurological disorders) the level of NO is
elevated as a result of over-activation of NMDA receptor in neurons or iNOS activation in glial
cells. At this high concentration, NO can reacts with superoxide anion to form the very reactive
peroxynitrite that causes neuronal toxicity. NO has also been shown to cause S-nitrosylation of
2

several proteins that is responsible for neuronal death (Blaise et al., 2005; He et al., 2007; Cho et al.,
2009). Thus, whilst NO plays a physiological role in neuronal cell signaling, its over-production
may cause neuronal death.
Figure 1. Schematic drawing of the NO signaling cascade. The enzyme nitric oxide synthase (NOS)
is stimulated by calcium–calmodulin (Ca/CaM). In the presence of O2 and NADPH NO is formed.
NO binds to the heme-moiety of soluble guanylyl cyclase (sGC) resulting in the stimulation of the
enzyme which result in elevation of cGMP level.
1.2. NO and early stages of CNS development
1.2.1. Neuronal precursor proliferation
The complex mammalian CNS, with its diverse cell types and billions of connections, undergoes
several cellular stages of development. The formation of the CNS begins early in development with
the induction of the neural ectoderm on the dorsal surface of the embryo. Subsequently, the neural
ectoderm plate changes its shape to form a neural groove and eventually, a neural tube. The wall of
the neural tube is composed of germinal cells, collectively called the neuroepithelium, which
produces neurons and glia throughout the CNS (Wessley and De Robertis, 2002; Spitzer, 2006;
Corbin et al., 2008). After closure of the neural tube at an early embryonic stage, process of
neuronal proliferation, migration and differentiation occurs sequentially but partially overlapping
(Spitzer, 2006; Ayala et al., 2007).
During development, transient expression of nNOS has been demonstrated in different brain areas,
which implicate functional role of NO in embryonic neural tissue formation (Bredt and Snyder,
3

1994; Santacana et al., 1998; Chen et al., 2004; Nott and Riccio, 2009). In line with this, NO has
been shown to suppress the proliferation of neural precursor cells in developing Drosophila
imaginal disks (Kuzin et al., 1996; Enikolopov et al., 1999), Xenopus tadpole optic tectum
(Peunova et al., 2001, Peunova et al., 2007) and cerebellar granule cells of new born rats (Ciani et
al., 2006). In developing chicken neural tube NO is endogenously produced which has been
proposed to regulate cell-cycle progression. It has been demonstrated that high NO levels promoted
entry into S phase basally, whereas low levels of NO facilitated entry into mitosis apically (Traister
et al., 2002). Moreover, in cultured human neuroblastoma cell lines NO has been showed to inhibit
neuronal precursor cell proliferation (Murillo-Carretero et al, 2002; Ciani et al., 2004). Treatment
of dissociated mouse cortical neuroepithelial cluster cell cultures and rat cerebellar granule cells
with NOS inhibitor or scavenging of NO resulted in enhanced proliferation (Cheng et al., 2003;
Ciani et al., 2004). These studies show the anti-proliferative role of endogenous NO in neuronal
precursor cells; however, the mechanism is not fully understood. In developing Xenopus, cerebellar
granule cells and human neuroblastoma cells, the cGMP/PKG pathway has been suggested to
mediate the anti-proliferative role of NO (Peunova et al., 2007; Ciani et al., 2004; Ciani et al.,
2006). In both cerebellar cells and neuroblastoma culture the oncogenic transcription factor, N-
Myc, has been shown to be over expressed due to down regulation of cGMP/PKG pathway
(Contestabile A., 2008). On the contrary, other studies demonstrated that the anti-proliferative effect
of NO is cGMP independent (Phung et al., 1999; Young et al., 2000; Gibbs, 2003).
1.2.2. Neuronal migration
Neurons and neuronal precursor cell migrate long distances along the dorsal-ventral and anterior-
posterior axes of the nervous system during the early embryonic period, which is important for the
formation of functional neuronal network. Two modes of neuronal migration has been identified:
radial migration, in which cells migrate from the progenitor zone toward the surface of the brain
following the radial layout of the neural tube; and tangential migration, in which cells migrate
orthogonally to the direction of radial migration (Hatten, 1999; Marin and Rubenstein, 2003; Ayala
et al., 2007; Metin et al., 2008). In the developing rat brain, nNOS reaches the highest expression
level between embryonic day 16 and postnatal day 0, and this expression corresponds with the
migration of neuronal precursors from the ventricular zone to the external layers of the cortex
(Bredt and Snyder, 1994; Nott et al., 2008).
The first functional evidence on the role of NO in neuronal migration came from study conducted
on migrating granule cells of rat (Tanaka et al., 1994). The migration of cerebellar granule cells was
decreased in the presence of NOS inhibitor or haemoglobin that scavenges NO (Tanaka et al.,
4

1994). In embryonic grasshopper NO has been demonstrated to be essential in enteric neuron
migration (Haase and Bicker, 2003; Knipp and Bicker, 2009). They showed that pharmacological
inhibition of NOS or sGC blocks neuronal migration, whereas the blocking can be rescued by
applying sGC activators or cGMP analogue suggesting positive regulatory role of NO/cGMP
signaling in neuronal migration. In developing Xenopus, NO has been shown to facilitate neuronal
cell migration (Peunova et al., 2007). Recent studies using genetic approaches indicated that nNOS
is essential for cortical development (Nott et al., 2008; 2009). Here, radial migration of cortical
progenitors was impaired in nNOS null mice. Moreover, ex vivo electroporation of the mutant, NOinsensitive
histone deacetylase 2 (HDAC2) in cortical progenitors impaired radial migration
indicating that NO positively regulates neuronal migration via S-nitrosylation (Nott et al., 2009). In
the fetal human spinal cord, some neurons expressed NOS as they migrate to their final destination,
which suggest that NO may involve in neuronal migration during human nerve cell development
(Foster and Phelps, 2000).
1.2.3. Neuronal differentiation
Neuronal differentiation has many components, which includes maturation of electrical excitability,
neurotransmitter specification, outgrowth of axon and dendrites (Spitzer, 2006). Evidence
supporting that NO mediate neuronal differentiation came from nerve growth factor (NGF) induced
differentiating PC12 cells (Pennova and Enikolopov, 1995). In these cells the expression of the
three isoform of NOS corresponds with NGF induced neuronal differentiation. Moreover,
application of NOS inhibitor in PC12 culture increased cell proliferation but inhibit differentiation
into neurons. Subsequent studies indicated that NO induce differentiation into neuronal phenotypes
while inhibiting cell proliferation in various models (Ghigo et al., 1998; Phung et al., 1999; Cheng,
2003; Ciani et al., 2004; Ciani et al., 2006, Evangelopoulos et al., 2010). The induction of NOS
expression in response to NGF has been shown to be mediated through a combination of the
neurotrophic tyrosine kinase receptor type 1 (TrkA) and Ras-Erk members of the mitogen activated
protein kinase (MAP kinase) signaling cascade (Bulseco et al., 2001; Schonhoff et al., 2001).
Neuronal growth cones, specialized fan-shaped structures at the tips of growing neurites, are
important for axon elongation and guidance. Filopodia, rod-like projections from growth cones,
responds to various extracellular cues during neurite elogation and guidance (Goldberg and
Burmeister, 1989; Kater and Rehder, 1995). During the early development of primary
somatosensory neurons, nNOS is expressed at time of neurite extension (Thippeswamy and Morris,
2002). Several in vitro studies demonstrated that growth cones of rat dorsal root ganglion (DRG),
Xenopus and chick retinal ganglion and Helisoma buccal ganglion collapsed in the presence of high
5

concentration of NO (Hess et al., 1993; Renteria and Constantine-Paton, 1996; Ernst et al., 2000;
He et al., 2002; Trimm and Rehder, 2004). Recently, S-nitrosylation of microtubule-associated
protein 1B (MAP1B) has been suggested to mediate NO-induced axon retraction in cultured
vertebrate neurons (Stroissnigg et al., 2007). In Helisoma buccal ganglion, NO regulates growth
cone filopodial behavior via sGC, PKG and cyclic adenosine diphosphate ribose (cADPR), which
causes the release of calcium from intracellular stores via the ryanodine receptor (RyRs)
(Welshhans and Rehder, 2005). The downstream effector proteins for NO/cGMP signaling, PKG
(Yue et al., 2008) and CNGs (Togashi et al., 2008) have been shown to mediate ephrin-A5-induced
growth cone collapse and Sema3A-induced growth cone repulsion, respectively. Furthermore, the
NO/cGMP signaling has been shown to negatively regulate the Ca 2+ -induced Ca 2+ release (CICR)
through RyRs to control directional polarity of DRG axon guidance (Tojima et al., 2009). Here, a
Ca 2+ signal produced by photolysing caged Ca 2+ caused growth cone repulsion on laminin substrate
which was converted into attraction by pharmacological blocking of NO/cGMP pathway or genetic
deletion of nNOS.
On the other hand, studies performed in vitro on PC12 cells (Hindley et al., 1997; Rialas et al, 2000;
Yamazaki et al., 2001) and neuroblastoma cells (Evangelopoulos et al., 2010) indicated that NO
increases neurite outgrowth. In developing antenna of the grasshopper embryo where two siblings
of pioneer neurons establish the first two axonal pathways to the CNS, NO/cGMP signaling has
been shown to mediate axonogenesis (Seidel and Bicker, 2000). Here, pharmacological inhibition
of NOS and sGC resulted in abnormal pattern of pathfinding, loss of axon emergence and axon
retraction suggesting that NO/cGMP signaling is a positive regulator of neurite elongation. Recent
study indicated that S-nitrosylation HDAC2 caused by neurotrophin induced NO signaling is
necessary for dendritic outgrowth of embryonic cortical neurons (Nott et al., 2008). In models of
CNS injury, the regeneration of axons in embryonic insect (Stern and Bicker, 2008) and optic nerve
in the goldfish (Koriyama et al., 2009) was facilitated by NO/cGMP signaling pathway.
1.2.4. Synaptogenesis
Synaptogenesis can be defined as the assembly of pre- and postsynaptic proteins into the highly
specific structure of the synapse. These major components of glutamatergic synapses includes;
synaptic vesicles (SVs), glutamate receptors, active zone proteins, postsynaptic density (PSD)
scaffolding proteins, and trans-synaptic adhesion molecules. The pre-and postsynaptic components
of a synapse must accumulate at sites of physical contact between axons and dendrites with precise
timing (McAllister, 2007). During development, synaptogenesis is tightly coupled to neuronal
differentiation and formation of neuronal circuitry. For example, shortly after neurons differentiate
6

and extend axonal and dendritic processes, many of the genes encoding synaptic proteins are turned
on, resulting in the formation, accumulation, and directional trafficking of vesicles carrying pre- and
postsynaptic protein complexes. During this time, the specification of correct neuronal connections
is determined, as axons and dendrites make contact and establish initial, often transient, synapses.
Thus, the process of synaptogenesis involves a myriad of secreted factors, receptors, and signaling
molecules that make neurons receptive to form synapses (Waites et al., 2005).
Transient expression of NOS has been suggested to correspond with the period of active synapse
formation (Williams et al., 1994; Ogilvie et al., 1995; Sporns and Jenkinson, 1997; Gibbs and
Truman, 1998; Bicker, 2005). Moreover, in embryonic grasshoppers, synaptogenesis correlates with
a phase when many identifiable nerve cell types respond to NO by producing cGMP (Truman et al.,
1996; Ball and Truman, 1998). The dynamic regulation of NO-induced cGMP formation during
synaptic maturation has been also described in the embryonic grasshopper brain and at the larval
neuromuscular junction of Drosophila (Wildemann and Bicker, 1999; Seidel and Bicker, 2002). NO
signaling has been implicated to play an important role in activity-dependent synaptic maturation
(Nikonenko et al., 2003). Here, inhibition of NOS has been demonstrated to interfere with calciumdependent
growth of filopodia-like outgrowth and presynaptic remodelling of varicosites while NO
donors could facilitate them.
Excitatory synapse formation, elimination and remodelling on dendritic spines represent a
continuous process, which shapes the organization of synaptic networks during development
(Yoshihara et al., 2009). NO is produced at excitatory synapses by nNOS which is localized to the
synapse through its interaction with the second PDZ domain of PSD-95. Overexpression of PSD-95
resulted in the rise of nNOS, leading to the formation of spines which became innervated by
multiple presynaptic partners (Nikonenko et al., 2008). A similar observation was made when the
concentration of NO is increased in the tissue by exogenous application of a NO donor. Conversely,
blockade of nNOS interferes with synapse formation and resulted in a loss of spines. NO produced
by expression of the PSD in the newly formed spine could thus represent a retrograde signal
stimulating nearby axons to differentiate and form a presynaptic terminal (Nikonenko et al., 2008;
Yoshihara et al., 2009).
1.3. NO and adult neurogenesis
The dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) possess the capacity
to generate new neurons in adult animals (Gage, 2000; Curtis et al., 2003). The whole proliferation
area in both SVZ and DG has been suggested to be under the influence of NO (Estrada and Murillo-
Carretero, 2005). Neuroanatomical studies indicate that nNOS and sGC is expressed in adult SVZ
(Moreno-López et al., 2000; Gutierrez-Mecinas et al., 2007). The existing evidence from several
7

laboratories implicate that NO exerts a negative control on proliferation of cells in SVZ (Packer et
al., 2003; Cheng et al., 2003; Moreno-Lopez et al., 2004).
Reports on the role of NO in adult hippocampal neurogenesis; however, are contradictory. Systemic
application of NOS inhibitors, nitro-L-arginine methyl ester (L-NAME) or 7- nitroindazole (7NI) to
adult rodents did not alter the number of mitotic cells in the DG of the hippocampus (Moreno-
Lopez et al., 2004) but the density of Bromo-deoxyuridine (BrdU) positive cells increased
significantly after chronic inhibition of NOS ( Park et al., 2003). nNOS derived NO has been shown
to inhibit adult neurogenesis in DG by down-regulating cyclic AMP response element binding
protein (CREB ) phosphorylation (Zhu, 2006). The use of mutant mice lacking functional nNOS
resulted in increased proliferation in DG (Packer et al., 2003) whereas mice lacking eNOS showed a
significant reduction in neuronal progenitor cell proliferation in the same structure (Reif et al.,
2004). NSC proliferation and survival of new born neurons in the hippocampus were investigated in
nNOS knock out mice and double knock out mice (nNOS/eNOS). The proliferation of NSC was not
significantly altered in nNOS knock out mice but survival of newly formed neurons was
substantially higher (Fritzen et al., 2007). In contrast, nNOS/eNOS double knockout mice had
significantly decreased survival rates.
Brain injuries such as ischemia, traumas and seizures are characterized by overproduction of NO.
This could be as a result of nNOS activation following massive release of glutamate, up-regulation
of nNOS and iNOS (Estrada and Murillo-Carretero, 2005). It has been demonstrated that expression
of iNOS is necessary for ischemia-stimulated cell birth in the DG after focal cerebral ischemia (Zhu
et al., 2003; Luo et al., 2005). Similarly, exogenous NO source reported to increase cell
proliferation and neuroblast migration in SVZ and DG (Zhang et al., 2001; Chen et al., 2004; Cui et
al., 2009). In contrast, decreased nNOS expression was observed in SVZ which corresponds to
increased cell proliferation but reduced cell migration after focal cerebral ischemia in rats (Sun et
al., 2005; Zhang et al., 2007). A recent study demonstrated that cerebral ischemia reduces
hippocampal nNOS expression, and inhibition of nNOS ameliorates ischemic injury, stimulates cell
proliferation, and up-regulates iNOS expression and CREB phosphorylation (Luo et al., 2007).
Thus, it appears that the source of NO (whether it is from eNOS, iNOS or nNOS) and condition of
the animal (health versus disease) dictate the role of NO in adult neurogenesis.
1.4. NO signaling in neurodegenerative diseases
Since NO is an important regulator of nervous system functions, substantial changes in NO and
cGMP synthesis may lead to nervous system degeneration. Several studies have indicated that NO
may directly or indirectly play a key role in the pathogenesis of a number of neurodegenerative
8

diseases including Alzheimers, Amyotrophic lateral sclerosis (ALS) and Parkinson diseases
(Madhusoodanan and Murad, 2007).
The role of NO in the development of Parkinson disease has been extensively studied in 1-methyl-
4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) induced animal
models. Previous studies implicated that mice lacking nNOS are more resistant to MPTP induced
neurotoxicity (Przedborski et al., 1996; Matthews et al., 1997). The nNOS inhibitor 7NI has been
shown to protect against MPTP-induced neurotoxicity in experimental animals (Kurosaki et al.,
2002; Watanabe et al., 2008). 7NI has been also showed to protect from 6-OHDA induced
neurodegeneration whereas NO donor worsened the neurodegeneration (Di Matteo et al., 2009).
The majority of studies indicated cGMP independent mechanism of action of NO in mediating
MPTP induced neurotoxicity. These cGMP-independent actions of NO include oxidation of thiols,
S-nitrosylation and nitration of proteins (Madhusoodanan and Murad, 2007).
In Alzheimer’s disease (AD) all the three isoforms of NOS have been shown to be elevated,
indicating an important role for NO in the pathomechanism of AD (Thorns et al., 1998; Lüth 2001).
Nitrotyrosine which has been found in AD patients was highly co-localized with nNOS in cortical
pyramidal cells (Simic et al., 2000; Sultana et al., 2006). Immunocytochemical methods
demonstrated that the nNOS, iNOS and nitrotyrosine immunopositive cell bodies over the entire
chronic AD patients brains (Fernández-Vizarra et al., 2004).
Since the expression and activity of nNOS is increased in many CNS diseases, the NO/cGMP
signaling pathway could be a putative target for developing drugs. In line with this, several nNOS
inhibitors were developed but appear to have side effects (Zhou and Zhu, 2009). To minimize
unwanted effects in recent years more attention is given toward designing a selective nNOS
inhibitor with a potential implication to target neurodegenerative diseases (Silverman, 2009). On
the other hand NO synthesized by eNOS has been shown to be neuroprotective in an in vitro model
of ALS (De Palma et al., 2008). Here, overexpression of eNOS by neurons has been suggested as a
neuroprotective mechanism in neurodegenerative diseases. Changes in histone acetylation that
certainly involve NO signaling have been implicated to partially improve memory loss and
neurodegeneration (Fischer et al., 2007, Riccio et al., 2006, Nott et al., 2008). Thus, careful
manipulation of NO signaling may prove to be a potential therapeutic approach in
neurodegenerative disorders.
1.5. Human neuronal stem cells as a model of developing nerve cells
Stem cells are defined as precursor cells that have the ability to self-renew and to generate various
differentiating cell types. Pluripotent stem cells can give rise theoretically to every cell type in the
animal body including neurons. Three types of mammalian pluripotent stem cell lines have been
9

isolated: embryonal carcinoma (EC) cells, the stem cells of testicular tumours; embryonic stem (ES)
cells derived from pre-implantation embryos; and embryonic germ (EG) cells derived from
primordial germ cells (PGCs) of the post-implantation embryo (Donovan and Gearhart, 2001).
Neuronal stem cells (NSC) are pluripotent cells that have the ability to generate the three major cell
types of the CNS; neurons, astrocytes and oligodendrocytes. The search for a therapeutic option to
treat neurodegenerative diseases caused an expansion of NSC biology, which revealed numerous
transcription factors, growth factors and signaling pathways involved in development and
regeneration of CNS (Gage, 2000; Curtis et al., 2003; Jin et al., 2004; Shan et al., 2006; Boucherie
and Hermans, 2009).
Neuronal stem cells could be employed as important tool to dissect the development of human
nervous system. For example, several of the existing human EC stem cell lines provide robust and
simple culture systems to study certain aspects of cellular differentiation that mimic vertebrate
neurogenesis (Przyborski et al., 2004). The teratocarcinoma cell line Ntera2 (NT2) which is
derived from a human testicular cancer can be induced to differentiate into fully functional
postmitotic neurons and other cell types of the neuronal lineages (Andrews 1984; Pleasure et al.,
1992; Paquet-Durand and Bicker, 2007). NT2 cells shares many similarity with human embryonic
stem cells (Schwartz et al., 2005) and differentiation of NT2 cells into neurons has been suggested
to resemble vertebrate neurogenesis (Przyborski et al., 2000; Houldsworth et al., 2002; Przyborski
et al. 2003). Moreover, fully differentiated NT2 neurons have been shown to express a variety of
neurotransmitters in vitro (Guillemain et al., 2000; Podrygajlo et al., 2009) and form functional
synapse (Hartley et al., 1998; Podrygajlo et al., 2010).
More restricted stem cells with developmental potential can also be obtained from a variety of
tissues at different stages of development, including mature tissues from adult and fetal organisms.
For example, fetal human neural progenitor cells (hNPCs) that have the capacity to give neurons,
astrocytes and oligodendrocytes have been shown to mimic early developing human brain with
respect to cell proliferation, migration and differentiation (Fritsche et al., 2005; Moors et al., 2007;
Moors et al., 2009). NSCs also exist within limited regions of the adult CNS (SVZ and
hippocampus), and it is possible to isolate and expand these cells to give rise to progenitor cells
restricted to defined neural lineages, neuronal and glial cells (Gage, 2000).
Another promising model for developing human neurons is induced pluripotent stem cells (iPS)
obtained by molecular reprogramming of somatic cells. These cells have been successfully
differentiated into dopaminergic neurons and functionally integrated into rat brain (Werning et al.,
2008).
10

1.6. Hypothesis and aim of the study
The enzymes that synthesize NO and its target enzyme soluble guanylyl cyclase have been shown
to be expressed during development of nervous system in both vertebrate and invertebrate.
Functional studies that include neuronal proliferation, migration and synaptogenesis strongly
suggest that NO signaling is involved in the early development of the nervous system. However, it
is unknown whether NO signaling plays a functional role in early developing human nerve cells.
Since most of the molecules involved in neuronal migration, axon guidance and synaptogenesis are
conserved and some neurons in fetal human spinal cord express NOS as they migrate to their final
destination, we have hypothesized that NO may play functional role to modulate neuronal precursor
migration and synaptic maturation in developing human brain. To test the hypothesis I have used
two models of stem cell derived human neuronal culture. The NT2 spherical aggregates and fully
differentiated NT2 neurons which are obtained from human teratocarcinoma cell line (Ntera2).
Additionally, I have used fetal human neural progenitor cells (hNPCs) which was maintained as
free-floating three-dimensional neurosphere primary culture.
The aim of this work was to test whether the gaseous messenger NO modulates the proliferation,
migration, differentiation and pre-synaptic release in developing human model neurons. Specifically
we tested the following hypothesis:
1. The motility of cells from human neuronal precursor (NT2) cells spherical aggregate is
regulated by NO and cGMP signal transduction during differentiation into neuronal
phenotypes.
2. The gaseous messenger NO and cyclic nucleotide signaling induces pre-synaptic vesicle
release in developing human model neurons.
3. The migration of cells from fetal human neural progenitor cells is regulated by NO
signaling.
11

2. Discussion
2.1. Nitric oxide and cGMP signal transduction positively regulates the motility of human
neuronal precursor (NT2) cells
Neuronal migration is a critical event during early development of the nervous system. Human
neurological disorders such as lissencephaly, cortical heterotopias and microcephaly are associated
with defective neuronal migration (Ayala R et al., 2007; Mètin C., et al., 2009). During
development, neurons and neuronal precursor cells migrate long distances in response to specific
external signals. This coordinated movement is subjected to modulation by intra-and intercellular
signals. The gaseous messenger NO has been implicated to regulate neuronal migration in both
vertebrates and invertebrates (Tanaka et al., 1994; Haase and Bicker, 2003; Peunova et al., 2007;
Knipp and Bicker, 2009). In this study we showed for the first time that the NO/cGMP/PKG
signaling pathway facilitates the migration of human neuronal precursor (NT2) cells. The NT2 cells
which were cultured as three-dimensional free-floating spherical aggregate exhibit many feature of
developing brain cells. Firstly, NT2 cell spheres express both precursor (nestin) and early neuronal
marker (�III-tubulin) and incorporate BrdU. Upon culturing on adhesive substrates such as
Matrigel, cells migrate out of the NT2 cell spheres, elaborate neuronal processes and differentiate
into fully functional neurons (Tegenge and Bicker, 2009; Tegenge et al., 2009).
Subpopulations of the migrated cells and cells within the NT2 aggregate express NO-sensitive sGC
that synthesize increasing level of cGMP (Fig.4, Tegenge and Bicker, 2009). Application of enzyme
inhibitors of nNOS, sGC and PKG blocked the migration of cells out of NT2 aggregates. Whereas
the blocking of migration by sGC inhibitor was reversed in the presence of a cell membrane
permeable analogue of cGMP. These results strongly indicate that the NO/cGMP/PKG signaling
pathway is essential for the migration of human neuronal precursor cells. Moreover, exogenous use
of NO and membrane permeable analogue of cGMP facilitated cell motility. Although sGC and
PKG was identified to act downstream of NO, I did not examine how the NO activated cGMP and
PKG modulate neuronal migration. Since one common feature among signaling pathways that
regulate neuronal migration is the eventual involvement of the cytoskeleton, I predict that NO
activated cGMP/PKG pathway may cause reorganization of actin. The reorganization of actin
cytoskeleton in response to cGMP/PKG that involves RhoA GTPase and phosphorylation of
Enabled/vasodilator-stimulated phosphoprotein family proteins has been reported (Sporbert et al.,
1999; Gudi et al., 2002; Borán and García 2007; Lindsay et al., 2007, Zulauf et al., 2009).
Since several studies implicated that NO inhibit neuronal precursor cells proliferation while
enhancing neuronal differentiation (Ghigo et al., 1998; Phung et al., 1999; Cheng, 2003; Ciani et
12

al., 2004; 2006), I tested the effect of chemicals on neuronal proliferation and differentiation. Here,
concentration of both activators and inhibitors of small bioactive chemicals used to modulate cell
migration did not significantly affect cell viability, proliferation and neuronal differentiation
indicating that the observed effect was mainly on cell migration. However, the NO donor (100 µM
NOC-18) differentially coordinated cell motility and proliferation. At this concentration, NOC-18
seemed to facilitate cell migration while inhibiting proliferation (Fig. 6, Tegenge and Bicker, 2009).
This effect of NOC-18 on neuronal progenitor proliferation is in agreement with several studies that
showed anti-proliferative effect of NO (Enikolopov et al., 1999; Murillo-Carretero et al, 2002;
Ciani et al., 2004;Peunova et al., 2007). Moreover, we observed that under inhibition of nNOS
there was a modest decrease in the percentage of neuronal cells while the cGMP analogue slightly
increased differentiation into neurons (Tegenge and Bicker, 2009). Thus, the present data show
concentration dependent effects of NO/cGMP signaling on the distinct process of neuronal
proliferation, migration and differentiation which could account partly for the reported
inconsistency on the role of NO at early stage of nervous system development.
In summary, this study presented a model of human neuronal precursor cells as three-dimensional
spherical aggregates that proliferate, migrate and differentiate into neurons. The migration of cells
from NT2 spherical aggregates is modulated by NO/cGMP/PKG signaling pathway. Since NT2
cells spherical aggregates elaborate neuronal process, it will be interesting to investigate the effect
of NO on the growth cone response and neurite outgrowth.
2.2. Nitric oxide and cyclic nucleotide signal transduction modulates synaptic vesicle turnover
in human model neurons
The human embryonal carcinoma stem cell line Ntera-2/cl.D1 (NT2), is extensively studied as a
model of human neuronal differentiation in vitro. The NT2 cells have been demonstrated to
terminally differentiate into functional postmitotic neurons in vitro (Andrews, 1984; Pleasure et al.,
1992; Paquet-Durand and Bicker 2007). The major drawback was the rather lengthy differentiation
protocol, which has been shortened significantly in recent years by employing the aggregate culture
method (Paquet-Durand et al., 2003; Podrygajlo et al., 2009). In this study we followed the
synaptic maturation of human NT2 neurons generated by this novel culture method. Human NT2
neurons express the dendritic marker (MAP2) and a marker of axogenesis (phosphorylated Tau).
While MAP2 is expressed in short process at all stages of NT2 neurons, the staining for Tau on
longer neurites appeared only later upon culturing NT2 neurons (about 4 weeks).
During synaptogenesis the amount of both pre- and postsynaptic proteins increases considerably
(McAllister A.K, 2007). Here, synaptic maturation was followed by immunocytochemical staining
13

of the pre-synaptic vesicle associated proteins (synapsin I and synaptotagmin I). The staining of
both proteins appeared as punctate along the neurites and increased significantly within 2-4 weeks
of in vitro culture indicative of pre-synaptic maturation (Figs 1&2, Tegenge et al., 2009). Evidence
for activity-dependent pre-synaptic vesicle release was provided by employing antibody against the
luminal domain of synaptotagmin I. Upon fusion of synaptic vesicles with the plasma membrane,
the luminal domain of synaptotagmin becomes exposed to the neuronal surface, allowing the
binding of the antibody and their subsequent internalization through the endocytosis of synaptic
vesicle (Figure 2a). In human NT2 neurons I demonstrated that luminal synaptotagmin antibody is
incorporated in both depolarization and calcium dependent manner, which suggest that the neurons
display synaptic vesicle recycling. In NT2 neurons cultured only for 1 week we hardly detected
luminal synaptotagmin punctate staining compared to intense staining within 2-4 weeks. Thus,
activity-dependent incorporation of luminal synaptotagmin antibody depends on the length of in
vitro culture of the neurons indicating that human NT2 neurons undergo synaptic maturation
(Tegenge et al., 2009).
Figure 2. Methods to monitor the synaptic vesicle recycling and exocytosis in developing neurons.
(a) Use of antibodies against the luminal domain of the synaptic vesicle protein synaptotagmin (Syt-
14

ecto abs). Upon fusion of synaptic vesicles with the plasma membrane, the luminal domain of
synaptotagmin becomes exposed to the neuronal surface, allowing the binding of Syt-ecto abs and
their subsequent internalization through synaptic-vesicle endocytosis. (b) Use of the amphiphilic
dye FM1–43, which very effectively labels endocytic vesicles (loading and wash) and dissociates
from membranes upon re-exposure of the vesicle lumen to the extracellular medium during
exocytosis (unloading). Adopted from (Matteoli et al., 2004).
Independently, a second functional evidence for human NT2 neurons was provided by imaging
synaptic vesicle recycling and exocytosis employing fluorescent stryl dye, FM1-43. The dye labels
synaptic vesicle that undergo vesicle recycling and dissociates from the vesicles to the extracellular
medium upon a second round of exocytosis (Figure 2b). FM1-43 imaging revealed that depolarized
NT2 neurons display synaptic vesicle recycling and exocytosis in the presence of calcium. This pre-
synaptic vesicle exocytosis induced by high K + presumably indicates neurotransmitter release at the
active zone of nerve terminals. Recently, our group has shown that in the absence of glial cells, NT2
neurons showed spontaneous synaptic currents and responded to exogenous application of GABA
and glutamate neurotransmitters (Podrygajlo et al., 2009, Podrygajlo et al., 2010). These results
suggest that GABA and glutamate are putatively released upon high K + stimulation. Thus, NT2
neurons represent pure human neuronal culture that displays rapid vesicle exocytosis. As a result,
NT2 neurons can be utilized as human model neurons to understand the cellular and molecular basis
of pre-synaptic vesicle release. Furthermore, time dependent synaptic maturation of NT2 neurons
can be exploited to screen chemical compounds influencing the expression of pre-synaptic proteins
and pre-synaptic release. In this regard NT2 neurons have greater potential as compared to the
widely used the neuroendocrine cell line PC12 cells (Westerink and Ewing, 2008) not only because
of its human origin but also due to its terminal differentiation into functional neurons.
Numerous studies demonstrated that NO induces pre-synaptic vesicle release (Hawkins et al., 1994;
Arancio et al., 1996; Meffert et al., 1996; Sporns and Jenkinson, 1997; Wildemann and Bicker,
1999; Li et al., 2002). Furthermore, NO/cGMP pathway has been shown to regulate synaptic vesicle
endocytosis and recycling by increasing pre-synaptic phosphatidylinositol 4, 5-bisphosphate (PIP2)
(Micheva et al. 2003; Petrov et al., 2008). In this study we showed for the first time that NO/cGMP
signaling cascade induces synaptic vesicle recycling and pre-synaptic release in human model
neurons. Firstly, expression of nNOS and NO-sensitive sGC was demonstrated suggesting
functional NO/cGMP signaling is present in human NT2 neurons. Previously, it has been shown
that expression of NO-sensitive sGC correlates with synaptogenesis (Truman et al, 1996; Ball and
Truman, 1998; Bicker, 2005). Secondly, two NO donors and membrane permeable cGMP analogue
facilitate synaptic vesicle recycling and vesicle exocytosis in human NT2 neurons (Tegenge et al.,
15

2009). In developing neurons synaptic vesicle turnover is important in the assembly of new synaptic
sites (Matteoli et al., 2004). Moreover, activation of NO signaling has been demonstrated to
facilitate activity-dependent pre-synaptic remodelling that contributes to synaptogenesis
(Nikonenko et al., 2003; 2008). Thus, induction of synaptic turnover by NO donors and cGMP
analogue in human NT2 neurons suggest that NO signaling may involve in activity-dependent
synaptic maturation of human nerve cells.
The data further demonstrated that the cAMP/PKA signaling induces synaptic vesicle turnover in
human NT2 neurons, which possibly involve phosporylation of synapsin. Phosphorylation of
synapsin by the cAMP/PKA signaling that leads to the release of vesicles from the reserve pool has
been suggested to enhance pre-synaptic vesicle releases (Fiumara et al., 2004; 2007; Bonanomi et
al., 2005; Menegon et al., 2006).
Taken together, I have shown that human NT2 neurons undergo pre-synaptic maturation in vitro.
Moreover, the release of neurotransmitter at pre-synaptic terminals of human NT2 neurons could be
modulated by NO and cyclic nucleotide signaling cascade. These result points toward critical role
of NO/cyclic nucleotide signaling as a regulator of neurotransmitter release in developing human
nerve cells.
2.3. Nitric oxide signaling as regulator of human neuronal progenitor cell migration
Previously, we demonstrated that NO/cGMP signaling facilitates the migration of cultured human
neuronal precursor (NT2) cells (Tegenge and Bicker, 2009). Since the NT2 cell line was obtained
from a human teratocarcinoma (Andrews, 1984) and we cannot rule out effects of genetic
rearrangements in the cancer cells, it is problematic to relate these findings to normal human neural
development. Multi-potent human neuronal progenitor cells can be obtained from cells of
ectodermal lineage and are restricted in fate to develop into neurons, astrocytes and
oligodendrocytes. Stem cells can also be isolated from both developing human brain and restricted
areas of adult human brain. Studies over the past decade have focused mainly on the use of stem
cells in transplantation therapy for various diseases models. However, stem cells derived neuronal
progenitor cells have the potential to investigate the molecular and cellular basis of human brain
development.
In this study, we used fetal human neural progenitor cells (hNPCs) cultured as neurospheres to
investigate the role of NO/cGMP signaling in neuronal migration. hNPCs derive from 16 to 20week
old female or male fetal whole brain homogenates, which have the capacity to differentiate
into neuronal and glial cells (Moors et al., 2009; Breier, 2009). Culturing proliferating human
neurospheres on laminin coated adhesive substrates resulted in cells that migrate out of the spheres
16

which were accompanied by differentiation into both neuronal and glial cells. Intriguingly, cells that
migrate out respond to exogenous stimulation of NO and synthesize increasing level of cGMP.
These results provide first experimental evidence that functional NO-sensitive sGC is expressed
early during human brain development. The cGMP positive cells at the forefront of migration were
co-stained for either nestin or GFAP but not for ����-tubulin. Thus, compared to NT2 cell culture
that lack GFAP positive cells, the hNPCs may mimic aspects of radial neuroblast migration during
human brain development.
Application of small bioactive enzyme inhibitors and activators in human neurosphere culture
implicate that NO/cGMP/PKG signaling is key regulator of neuronal progenitor migration.
Compared to the NT2 cell culture, in hNPCs inverted U-shaped dose-response curve for the NO
donor NOC-18 was obtained at relatively smaller dose range (1-100 µM). Our data demonstrate that
at 10 µM NOC-18 appeared to facilitate neuronal progenitor cell migration while at relatively
higher concentration (100 µM) inhibition of cell migration was observed. These result suggest that
NO could play dual role on neuronal migration depending on the concentration. Such dual role of
NO on cell migration and neurite outgrowth that depends on concentration has been reported
previously (Elferink, and VanUffelen, 1996; Trimm and Rehder, 2004). Furthermore, we directly
showed the involvement of sGC and PKG in NO induced facilitation of hNPCs migration. Here, coapplication
of NO donor with enzyme inhibitors of sGC or PKG resulted in blocked of NO induced
facilitation of hNPCs migration suggesting that low concentration of NO facilitates neuronal
progenitor migration via sGC and PKG signaling pathway.
In summary, this study provides the first evidence for the involvement of NO signaling in human
neuronal development. Since neuronal migration is controlled by a complex combination of
chemical signals, it would be interesting to comprehend how the NO signal regulates neuronal
migration in combination with other known signals. Understanding the cellular and molecular
mechanisms of neuronal migration may offer hopes to develop new therapeutic agents for human
congential disorders associated with defective neuronal migration. Our findings have further
implications in adult neurogenesis. In recent years research in NSC biology focus on restoration of
damaged neural networks in the various neurodegenerative diseases by promoting endogenous
neurogenesis or transplantation of NSC. To this end, manipulation of NO/cGMP signaling may
facilitate migration and integration of endogenous or transplanted neuronal progenitor cells into the
existing neuronal network which could promote functional recovery in various neurodegenerative
diseases.
17

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29

Acknowledgments
I thank my supervisor Prof. Dr. Gerd Bicker for allowing me to work in an exciting project and for
his excellent guidance throughout my PhD project. During my stay in your laboratory I have
learned not only how to become a good researcher but also a mentor. Thank you very much!
I also extend my sincere gratitude to my advisory group Prof. Dr. Stephan Steinlechner and Prof.
Dr. Herbert Hildebrandt for the useful discussion, suggestion and guidance throughout my doctoral
process.
I thank Prof. Dr.med. Ellen Fritsche and Dr. Thomas Dino Rockel (Group of Molecular Toxicology,
Institut für Umweltmedizinische Forschung, Heinrich Heine-University, Dusseldorf, Germany) for
permitting me to work on fetal human neural progenitor cells and continuous collaboration
throughout this project.
I thank ZSN PhD commission specially Prof. Dr. Wolfgang Löscher, Prof. Dr. Wolfgang
Baumgartner and the coordination office (Dr. Dagmar Esser, Nadja Borsum and Manuela
Chambers) for continuous support throught my doctoral study. I also sincerely thank the Ministry
for Science and Culture of Lower Saxony for giving me the Georg-Christoph-Lichtenberg
scholarship throughout my PhD study.
My stay in cell biology group could not be as fruitful as it is without the support and pleasant
environment created by present and past colleagues in the lab. I thank Dr. Micheal Stern for his
constructive comments, suggestion and discussion. I thank Saime Tan for her excellent technical
support. I am also thankful to Dr. Sabine Knipp and Dr. Grzegorz Podrygajlo for useful discussion.
My thanks also go to Arne Pätschke, Andrea Gierse, Rene Eickhoff, Nicole Böger, Frank Roloff
and Christina Lorbeer.
Last but not least I thank my family; Lydia and Betania. Thank you very much Lydia for the love,
courage and support you gave me throughout this work. Betu you helped a lot by staying long in the
kindergarten. Thank you for your patience.
30

Summary
The complex mammalian central nervous system (CNS) undergoes sequential but partially
overlapping cellular developmental processes that include cell proliferation, migration,
differentiation and synaptogenesis. The gaseous messenger nitric oxide (NO) has been implicated to
regulate each of these early neuronal developmental processes. However, it is not known whether
NO signaling is involved in the development of human CNS. Here, I used human neuronal stem
cells as a model of developing nerve cells to investigate the potential role of NO signaling in
neuronal migration, differentiation and pre-synaptic vesicle release.
In the first part, a model of human neuronal precursor cells was established from embryonic
carcinoma stem cells (NT2 cells) as three-dimensional free-floating aggregate culture. Upon
retinoic acid treatment the NT2 spherical aggregates generate cells that proliferate, migrate and
differentiate into neuronal phenotypes which mimic cellular processes that occur during early
development of CNS. Interestingly, the migrating cells and cells inside the spheres respond to
exogenous stimulation with NO by synthesizing cyclic guanosine-monophosphate (cGMP),
indicating that the NT2 spherical aggregates express functional NO/cGMP signaling. By employing
small bioactive enzyme activators and inhibitors of neuronal nitric oxide synthase (nNOS), soluble
guanylyl cyclase (sGC) and protein kinase G (PKG), I showed that the NO/cGMP/PKG signaling
cascade is a positive regulator of the migration of human neuronal precursor cells. Moreover, antiproliferative
effect of NO was observed at relatively higher concentration of a NO donor. A modest
but none significant effect of NO/cGMP signaling on neuronal differentiation was also noted.
In the second part, pre-synaptic maturation of human NT2 neurons were followed after the cells
migrate out of NT2 spherical aggregates and form neuronal networks. Human NT2 neurons express
increasing levels of the axonal marker (Tau) and typical pre-synaptic proteins (synapsin and
synaptotagmin I) depending on the length of in vitro culture. By employing an antibody directed
against the luminal domain of synaptotagmin I and the fluorescent dye (FM1-43), I showed that
depolarized mature NT2 neurons display calcium-dependent exo-endocytotic synaptic vesicle
recycling. NT2 neurons express nNOS and a functional NO receptor, sGC. Next, I examined
whether NO signal transduction modulates synaptic vesicle turnover, a pathway that may contribute
to activity dependent synaptic maturation in human model neurons. Two NO donors (sodium
nitroprusside, and N-Ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino ethanamine)) and cGMP
analog (8-Br-cGMP) enhanced synaptic vesicle turnover while a sGC inhibitor blocked the effect of
NO donors. NO donors evoked vesicle exocytosis which was partially blocked by the sGC
inhibitor. The activator of adenylyl cyclase, forskolin, and a cAMP analog induced synaptic vesicle
recycling and exocytosis via a parallel acting protein kinase A pathway. These data suggest that
31

NO/cyclic nucleotide signaling pathways may facilitate pre-synaptic neurotransmitter release in
human brain.
Finally, I tested the presence and function of NO/cGMP signaling in fetal human neural progenitor
cells (hNPCs). Fetal hNPCs were cultured as three-dimensional neurospheres that proliferate,
migrate and differentiate into both neuronal and glial cells. The migrating cells from human
neurosphere express functional sGC that synthesize increasing level of cGMP upon activation with
NO. Application of enzyme inhibitors of sGC and PKG blocked the migration of cells out of
human neurospheres. Inhibition of sGC can be rescued by a membrane permeable analog of cGMP.
In gain of function experiments both NO donor and cGMP analog facilitate cell migration
suggesting that NO-cGMP signaling positively regulates hNPCs migration. These results provide
first experimental evidence for a role of NO/cGMP signal transduction as a regulator of cell
migration during early development of the human brain.
32

Zusammenfassung
Das komplexe Zentralnervensystem (ZNS) der Mammalia durchläuft aufeinander folgende, zum
Teil auch sich überlagernde, zelluläre Entwicklungsprozesse, zu denen Zellproliferation, Migration,
Differenzierung und Synaptogenese gehören. Der gasförmige Botenstoff Stickstoffmonoxid (NO)
ist mit der Regulation jeder dieser frühen neuronalen Entwicklungsprozesse in Zusammenhang
gebracht worden. Allerdings ist noch nicht bekannt, ob eine NO Signalübertragung an der
Entwicklung des humanen ZNS beteiligt ist. In der vorliegenden Arbeit habe ich humane neuronale
Stammzellen als Modell für Nervenzellen in der Entwicklung verwendet, um die potentielle Rolle
der NO-Signalisierung während der neuronalen Migration, Differenzierung und der präsynaptischen
Vesikel Entleerung zu untersuchen.
Im ersten Teil der vorliegenden Arbeit wurde ein Modell für humane neuronale Vorläufer Zellen
aus embryonalen Karzinom Stammzellen (NT2) als dreidimensionale, frei flottierende Zellaggregat-
Kultur etabliert. Durch die Behandlung mit Retinsäure generieren die sphärischen NT2-Aggregate
Zellen, die proliferieren, migrieren und sich zu neuronalen Phänotypen differenzieren. Dies imitiert
zelluläre Prozesse, die während der frühen Entwicklung des ZNS ablaufen. Interessanterweise
sprechen die migrierenden Zellen und die Zellen innerhalb der Sphären auf exogene Stimulierung
mit NO an, indem sie zyklisches Guanosinmonophosphat (cGMP) synthetisieren. Dies deutet darauf
hin, dass die kugeligen NT2-Aggregate eine funktionale NO/cGMP-Signalübertragung aufweisen.
Unter Verwendung von kleinen, bioaktiven Enzymaktivatoren und -inhibitoren der neuronalen
Stickstoffmonoxid Synthase (nNOS), der löslichen Guanylyl Zyklase (sGC) und der Proteinkinase
G (PKG) zeigte ich, daß die NO/cGMP/PKG-Signalkaskade ein positiver Regulator für die
Migration humaner neuronaler Vorläuferzellen ist. Desweiteren konnte ein anti-proliferativer Effekt
von NO bei relativ höheren Konzentrationen des NO-Donors beobachtet werden. Außerdem war ein
leichter, jedoch nicht signifikanter Effekt der NO/cGMP-Signalisierung auf die neuronale
Differenzierung zu bemerken.
Im zweiten Teil wurde die präsynaptische Reifung der humanen NT2-Neurone verfolgt, nachdem
diese aus den spärischen NT2-Aggregaten migriert sind und neuronale Netzwerke geformt haben.
Humane NT2-Neurone exprimieren ansteigende Spiegel eines axonalen Markers (Tau) und typische
präsynaptische Proteine (Synapsin und Synaptotagmin I), abhängig von der Dauer der in vitro
Kultur. Unter Verwendung eines Antikörpers der gegen die luminale Domäne von Synaptotagmin I
gerichtet ist und einem Fluoreszenzfarbstoff (FM1-43) zeigte ich, daß depolarisierte ausgereifte
NT2-Neurone ein Calcium-abhängiges, exo-endozytotisches Recycling der synaptischen Vesikel
aufweisen. NT2-Neurone expremieren nNOS und einen funktionalen NO-Rezeptor, sGC. Als
nächstes untersuchte ich, ob die NO Signaltransduktion den Turnover der synaptischen Vesikel
33

moduliert. Dieser Signalweg könnte zur aktivitätsabhängigen synaptischen Reifung in humanen
Modellneuronen beitragen. Zwei NO-Donoren (Natriumnitroprussid und N-Ethyl-2-(1-ethyl-2-
hydroxy-2-nitrosohydrazino ethanamine)) und ein cGMP-Analogon (8-Br-cGMP) förderten den
synaptischen Vesikel Turnover, während ein sGC-Inhibitor den NO-Donor Effekt blockierte. NO-
Donoren riefen eine Vesikel Exozytose hervor, die teilweise durch den sGC-Inhibitor blockiert
wurde. Forskolin, ein Aktivator der Adenylyl Zyklase, und ein cAMP-Analogon induzierten ein
Recycling von synaptischen Vesikeln und Exozytose über einen parallel agierenden Proteinkinase
A-Signalweg. Diese Ergebnisse deuten an, daß die NO/zyklisches Nukleotid-Signalwege die
präsynaptische Neurotransmitter Ausschüttung im humanen Gehirn fördern könnten.
Schließlich habe ich das Vorkommen und die Funktion einer NO/cGMP-Signalübertragung in
fötalen humanen neuronalen Vorläuferzellen (hNPCs) überprüft. Fötale hNPCs wurden als
dreidimensionale Neurosphären kultiviert, welche proliferieren, migrieren und zu neuronalen und
glialen Zellen differenzieren. Die migrierenden Zellen aus humanen Neurosphären exprimieren
funktionale sGCs, welche durch Aktivierung mit NO ansteigende cGMP-Spiegel synthetisieren. Die
Zugabe von Enzym-Inhibitoren von sGC oder PKG blockierten die Zellmigration aus den humanen
Neurosphären. Die Inhibierung der sGC konnte durch ein membranpermeables cGMP-Analogon
aufgehoben werden. Sowohl ein NO-Donor, als auch ein cGMP-Analogon förderte die
Zellmigration in gain-of-function Experimenten, was nahelegt, daß eine NO-cGMP-
Signalübertragung die Migration von hNPCs positiv reguliert. Diese Ergebnisse liefern die ersten
experimentellen Beweise für eine Rolle der NO/cGMP-Signaltransduktion als ein Regulator für die
Zellmigration während der frühen Entwicklung des humanen Gehirns.
34

JOURNAL OF NEUROCHEMISTRY | 2009 | 110 | 1828–1841 doi: 10.1111/j.1471-4159.2009.06279.x
, ,
*Division of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, Hannover, Germany
Center for Systems Neuroscience (ZSN), Hannover, Germany
Abstract
Developmental studies in both vertebrates and invertebrates
implicate an involvement of nitric oxide (NO) signaling in cell
proliferation, neuronal motility, and synaptic maturation.
However, it is unknown whether NO plays a role in the
development of the human nervous system. We used a
model of human neuronal precursor cells from a well-characterized
teratocarcinoma cell line (NT2). The precursor cells
proliferate during retinoic acid treatment as spherical aggregate
culture that stains for nestin and bIII-tubulin. Cells migrate
out of the aggregates to acquire fully differentiated
neuronal phenotypes. The cells express neuronal nitric oxide
synthase and soluble guanylyl cyclase (sGC), an enzyme
Migration of neuronal progenitors from defined proliferative
zones to their final location is a key event that underlies the
development, regeneration, and plasticity of the brain.
During early development, newly born neurons undergo
extensive migration to set up the ordered organization of the
CNS and PNS. In the adult brain, the subventricular zone
(SVZ) and the subgranular region of the hippocampal dentate
gyrus maintain the capacity of generating new neurons that
migrate out to reach their ultimate locations (Hatten 1999;
Gage 2000; Lambert de Rouvroit and Goffinet 2001;
Nadarajah et al. 2003; Zheng and Poo 2007). The molecular
mechanisms of neuronal migration involve a rearrangement
of cytoskeletal components in response to extracellular cues,
mediated by numerous intra- and intercellular signals
(reviewed by Ayala et al. 2007; Kawauchi and Hoshino
2008). The gaseous messenger nitric oxide (NO) and its main
target soluble guanylyl cyclase (sGC), an enzyme that
synthesizes cGMP have been implicated in neuronal development
including neurogenesis and neuronal migrations
(reviewed by Enikolopov et al. 1999; Jurado et al. 2004;
Cárdenas et al. 2005; Bicker 2005, 2007). NO signal
transduction has been shown to differentially regulate cell
proliferation and motility in early developing Xenopus
that synthesizes cGMP upon activation by NO. The migration
of the neuronal precursor cell is blocked by the use of nNOS,
sGC, and protein kinase G (PKG) inhibitors. Inhibition of
sGC can be rescued by a membrane permeable analog of
cGMP. In gain of function experiments the application of a
NO donor and cGMP analog facilitate cell migration. Our
results from the differentiating NT2 model neurons point towards
a vital role of the NO/cGMP/PKG signaling cascade
as positive regulator of cell migration in the developing
human brain.
Keywords: cell migration, cGMP, neural development, nitric
oxide, Ntera 2, protein kinases G.
J. Neurochem. (2009) 110, 1828–1841.
(Peunova et al. 2007). Several studies indicate that NO
enhances neuronal cell migration (Tanaka et al. 1994; Zhang
et al. 2001; Haase and Bicker 2003). The transient expression
of NOS during development has provided additional
evidence for an involvement of NO signaling in neuronal
migration. For example, certain neurons in the fetal human
spinal cord express a histochemical marker for NOS as they
migrate to their final destination, suggesting a functional role
of NO during this developmental process (Foster and Phelps
2000). The limited availability of fetal tissue will certainly
Received May 15, 2009; revised manuscript received July 3, 2009;
accepted July 3, 2009.
Address correspondence and reprint requests to Gerd Bicker, Division
of Cell Biology, Institute of Physiology, University of Veterinary Medicine
Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany.
E-mail: gerd.bicker@tiho-hannover.de
Abbrevations used: 7NI, 7-nitroindazole; 8-Br-cGMP, 8-bromo-cylic
guanosine-monophosphate; BrdU, 5-bromo-2¢-deoxyuridine; cGMP-IR,
cGMP immunoreactivity; DMEM, Dulbecco’s modified eagle medium;
nNOS, neuronal nitric oxide synthase; NO, nitric oxide; ODQ, 1H-
[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one; PBS, phosphate-buffered
saline; PDL, poly-D-lysine; PKG, protein kinase G; PTX, PBS containing
0.2% Triton X-100; RA, retinoic acid; sGC, soluble guanylyl
cyclase; SNP, sodium nitroprusside; SVZ, subventricular zone.
Ó 2009 The Authors
1828 Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841

estrict experimental approaches to unravel the function of
NO signaling in human brain development.
The human teratocarcinoma cell line Ntera2 (NT2) is
derived from testicular cancer that can be induced to
differentiate into fully functional, post-mitotic neurons
(Andrews 1984; Pleasure et al. 1992). Evidence from gene
profiling of NT2 cells treated with retinoic acid (RA)
indicates the similarity of NT2 neural differentiation to
vertebrate neurogenesis (Przyborski et al. 2000, 2003; Houldsworth
et al. 2002). Moreover, NT2 neurons have been
successfully transplanted in human stroke patients (Nelson
et al. 2002; Hara et al. 2008). Recent studies from our group
have shown that NT2 cells could be induced to differentiate
into post-mitotic neurons upon RA treatment in spherical
aggregate culture and express a number of neuronal markers
(Paquet-Durand et al. 2003; Podrygajlo et al. 2009).
In this study we used NT2 spherical aggregates as a model
of human neuronal precursor cells to elucidate the involvement
of NO-cGMP signaling in neuronal precursor cell
migration. The NT2 spherical aggregates were characterized
by immunocytochemical methods for the expression of
neuronal markers, the neuronal isoform of the NO synthesizing
enzyme, neuronal nitric oxide synthase (nNOS), and
its target enzyme, sGC. Application of enzyme inhibitors and
activators to the differentiating NT2 cell aggregate provides
firm evidence that the NO/cGMP/ protein kinase G (PKG)
signal transduction pathway positively regulates human
neuronal precursor cell migration.
Materials and methods
Materials
The NO donor, NOC-18 (2,2-(Hydroxynitrosohydrazino) bis-ethanamine]),
was purchased from Calbiochem (Darmstadt, Germany),
and 8-bromo-cylic guanosine-monophosphate (8-Br-cGMP) and RP
isomer of 8-Br-cGMP were purchased from Alexis Biochemicals
(Lörrach, Germany). All the antibodies and other substances were
obtained from Sigma (Taufkirchen, Germany) unless otherwise
noted.
Cell culture
NT2/D1 precursor cells were purchased from American Type Culture
Collection (ATTC, Manassas, VA, USA) and treated as indicated in
the ATTC instruction manual. NT2 cells were cultured as free floating
spherical aggregate as described by Paquet-Durand et al. (2003).
Briefly, the NT2 precursor cells (passages 24–32) were seeded in
80 mm, bacteriological grade Petri dishes (Greiner, Hamburg, FRG)
at a density of 5 · 10 6 cells/dish in 10 mL of Dulbecco’s modified
eagle medium (DMEM/F-12; Gibco–Invitrogen, Karlsruhe, Germany)
supplemented with 10% fetal bovine serum (Invitrogen,
Karlsruhe, Germany) for 24 h. A differentiation medium, DMEM
containing RA at a final concentration of 10 lM was used with
medium change every 2–3 days. One group of spherical aggregates
were cultured in a dish with RA-containing medium for 7 days
(1 week) and the other group for 14 days (2 weeks).
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841
NO signaling regulates neuronal precursor motility | 1829
Migration assay
The spherical aggregate culture generated in a Petri dish was seeded
into poly-D-lysine (PDL) and Matrigel (Becton-Dickinson, Bedford,
MA, USA) coated microdishes (Ibidi GmbH, Munich, Germany) or
12-mm cover glasses at a density of approximately 2–10 aggregates.
The culture was allowed to attach for 90 min in an incubator at
37°C/5% CO 2. NOC-18 was prepared in 10 mM NaOH as 100 mM
stock solution, 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)
dissolved in dimethylsulfoxide as 20 mM stock, 7-nitroindazole (7NI)
dissolved in dimethylsulfoxide as 200 mM stock, and 8-Br-cGMP
and RP-8-Br-cGMP were dissolved in the medium. DMEM-containing
RA and chemical compounds were added into the culture and
incubated at 37°C/5% CO 2 for 48 h. To determine the migration of
cells out of the spherical aggregate we acquired images at desired
times as described in Moors et al. (2007).
Immunocytochemistry
For immunocytochemical stainings, we either used spherical aggregate
cultures after 48 h of the migration assay or aggregates that were
mechanically dispersed into single cells. The cultures were fixed in
4% paraformaldehyde dissolved in phosphate-buffered saline (PBS,
10 mM sodium phosphate, 150 mM NaCl, pH 7.4) for 30 min. They
were permeabilized by washing three times for 5 min in PBS
containing 0.2% Triton X-100 (PTX). Blocking solution containing
PTX and 5% normal horse or rabbit serum was applied for 1 h. The
primary anti-bIII-tubulin (1 : 10000; Sigma) and anti-nestin
(1 : 400; Calbiochem) were diluted in PTX-containing blocking
solution and applied overnight at 4°C. Wells were washed three times
for 5 min in PTX and incubated with secondary biotinylated antibody
(Vector, Burlingame, CA, USA) for 1 h at 20°C. After three more
washing steps in PTX, the immunofluorescence was detected using
streptavidin-CY3 (Sigma) or streptavidin-Alexa Fluor 488 (Mobitec,
Göttingen, Germany). For nuclear counter-staining cells were
incubated with 4¢-6-diamidino-2-phenylindole at a concentration of
1 lg/mL for 5 min in PBS. After final washing steps in PBS,
preparations were mounted in 90/10% glycerol/PBS with 4% sodium
n-propyl-gallate as antifading agent and sealed with nail varnish.
For the detection of cGMP immunoreactivity (cGMP-IR),
spherical aggregates were pre-incubated for 20 min at 20°C with
1 mM sodium nitroprusside (SNP) as a NO donor, 20 lM YC-1 [3-
(50-Hydroxymethyl-20-furyl)-1-benzyl indazole] as an enhancer of
NO-induced activity of sGC, and 1 mM 3-isobutyl-1-methylxanthine
as phosphodiesterase inhibitor. Cultures were washed once
with PBS and then we followed the same staining procedures as
above with a blocking solution containing PTX and 5% normal
rabbit serum. The polyclonal sheep cGMP antiserum (1 : 10000; a
kind gift from Dr. J. de Vente, Maastricht University, Netherlands)
was used as primary antibody to detect the level of cGMP. To test
for the contribution of endogenous enzyme activity to cGMP levels,
we used 50 lM ODQ as a specific inhibitor of sGC.
Western blotting
Cells were homogenized in a ristocetin-induced platelet agglutination
lysis buffer and HALT Ò protease inhibitor cocktail (Pierce,
Rockford, IL, USA) for about 30 min. The cell lysates were
centrifuged at speed of 6000 g for 10 min and the protein level of
supernatant was estimated by the BCA TM protein assay kit (Pierce).
Subsequently, equal amount of protein (100 lg) for NT2 and

1830 | M. A. Tegenge and G. Bicker
NT2 + RA-treated samples were boiled for 3 min in 2· Laemmli
buffer and subjected to electrophoresis on 8% sodium dodecyl sulfate
acrylamide gel. The gel was blotted on polyvinylidene difloride
membrane at 4°C. The membrane was then blocked with 3% dried
milk powder in PBS for 2 h and incubated overnight at 4°C with a
monoclonal antibody against nNOS (Sigma) at 1 : 1000 dilution in
blocking solution. The biotinylated secondary antibody was applied
for 1 h. Bound antiserum was detected by a standard peroxidase
staining techniques using the Vectastain ABC Kit (Vector). After
reactivation with methanol, membranes were additionally stained for
anti-acetylated-a-tubulin diluted 1 : 10000 in blocking solution.
Proliferation assay
Cell proliferation was assessed by application of the thymidine
analog 5-bromo-2¢-deoxyuridine (BrdU), which was incorporated
into the DNA of dividing cells. Spherical aggregates were exposed
to chemicals for 48 h and then incubated with BrdU (50 lM) for
4 h. After fixation with 4% paraformaldehyde for 30 min, they were
incubated with 2 M HCl solution in PBS for 20 min. Preparations
were washed three times with PBS to completely remove the acid.
They were permeabilized by washing three times for 5 min in 0.2%
PTX and blocked for 1 h in 5% normal horse serum. Then, we
followed the same immunocytochemical staining procedures as
described in the Immunocytochemistry with the primary monoclonal
anti-BrdU (clone Bu-33, Sigma) 1 : 200 in blocking solution.
Cell viability/cytotoxicity assay
To monitor cytotoxic effects of the drugs, we used the Alamar Blue
viability assay (Trek Diagnostic Systems, East Grinstead, UK) and
the Live/Dead viability/cytotoxicity assay (Molecular Probes,
Eugene, OR, USA). For Alamar Blue viability assay the NT2
spherical aggregates were mechanically dispersed into single cells.
About 10000 cell/well were seeded into PDL and Matrigel-coated
96-well plate and allowed to attach for 90 min. RA and chemical
compounds containing medium were added and incubated for 48 h at
37°C/5% CO 2. After 48 h the medium was completely changed into
a cell culture medium containing 3% Alamar Blue. Fluorescence
intensity of the Alamar Blue was detected at excitation/emission
wavelength of 530/590 nm using a microplate reader (Infinite M200,
Tecan, Männedorf, Switzerland) after 4 h. The actual number of
living or dead cells was determined by incubating NT2 aggregates
with 4 lM of ethidium homodimer-1 (EthD-1) and 2 lM calcein in
PBS for 30 min at 37°C/5% CO 2. After incubation the cultures were
washed in PBS and viewed under microscope. The average numbers
of living and dead cells were calculated from at least 10 images of
two independent experiments and compared with live controls which
was normalized to 100% for each experiment.
Microscopy and data analysis
The morphological observation and migration assay were performed
on a Zeiss Axiovert 200 microscope (Zeiss, Göttingen, Germany)
equipped with a CoolSNAP digital camera. Immunofluorescence
was detected on a Zeiss Axiovert 25 microscope equipped with an
Axiocam 3900 digital camera. The acquired images were processed
using Adobe Photoshop (Adobe Systems GmbH, München,
Germany). The migration was quantified by measuring the distance
from the edge of the sphere to the furthest migrated cell bodies at
four different locations per spherical aggregate (Moors et al. 2007).
The data were presented as mean ± SEM of at least eight spherical
aggregate. Each experiment was repeated at least three times.
The percentage of nestin, bIII-tubulin, and BrdU were determined
dividing the number of positively stained cells by the total
number of cells. The data were presented as mean ± SEM of two
independent experiments. Statistical comparisons of controls versus
treatment were performed with the unpaired two-tailed Student’s
t-test. Levels of significance were indicated as *p < 0.05,
**p < 0.01, and ***p < 0.001.
Results
Migration of cells out of the NT2 spherical aggregates
depends on the length of RA treatment
After forming confluent monolayers NT2 precursor cells
were split and seeded into bacteriological dishes. The cells
formed a loose aggregate after 24 h of the proliferation step
followed by subsequent RA treatment for 2 days. Upon
further incubation with medium containing RA for 1 week
the aggregates became spherical in shape, stable, and
surrounded by fewer isolated cells. When the use of RA
was prolonged for 2 weeks, the aggregates attained a more
spherical shape and elaborated neuronal processes. Accordingly,
we classified the aggregates into two separate groups
which were based on age as 1 or 2 weeks cell spheres.
The aggregates were seeded into PDL and Matrigel-coated
microdishes or cover glasses containing a marker grid, which
allowed us to follow the migration of cells out of the same
sphere for 48 h. Cells started to detach radially from the rim of
1-week-old NT2 sphere after about 3–6 h and the migrated
distance increased with time (Fig. 1a–c). Cells migrated to a
lesser extent from 2-week-old NT2 spheres but elaborated
neuronal processes (Fig. 1d–f). Higher magnification of the
migrated area of 1-week-old spheres showed mostly single
cells devoid of neuronal processes (Fig. 1g). However, 2week-old
spheres elaborated long neuronal processes (Fig. 1h)
and in this case length of neurites was excluded from the
migration analysis. Accordingly, migratory cells derived from
1- and 2-week-old spheres cover, after 48 h, a distance of
171.0 ± 5.5 and 77.5 ± 2.1 lm, respectively (Fig. 1i). Thus,
the migration of cells out of the NT2 spheres depended on the
duration of the RA treatment in the Petri dishes.
NT2 spheres express both precursor and neuronal markers
To monitor the progress of neuronal differentiation within
the developing spherical aggregate, we performed immunocytochemical
staining against cytoskeletal markers of progenitor
cells and neurons. At the end of a 48 h lasting
migration experiment spheres were fixed and stained against
the neuronal progenitor marker nestin and an early marker of
neuronal differentiation, bIII-tubulin. After 1 week of RA
treatment the NT2 spheres expressed nestin and bIII-tubulin
(Fig. 2a and c). For quantification, the spheres were
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841

Fig. 1 Cells migrate out of NT2 spheres
that were seeded on PDL and Matrigelcoated
microdishes. Representative photomicrographs
of NT2 spheres treated with
10 lM RA for 1 week (a–c) and 2 week (d–
f). The white lines in c delineate distance of
migration from the rim of the sphere to the
furthest migrated cells. Arrow heads in (f)
show representative neuronal processes. A
higher magnification of the migrated cells
shows single cells that bear no or few
neurite in 1-week-old spheres (g) when
compared with 2-week-old spheres that
elaborated long neurites (h). The use of RA
for 2 weeks significantly decreased cell
migration as compared with 1-week-old
spheres (i). Scale bars: 100 lm
(***p < 0.001).
dispersed mechanically into single cells and stained
against both markers. While 10.3% of the cells stained
positively for bIII-tubulin (Fig. 2e and h), about 84.3%
remained nestin-positive (Fig. 2g). Upon further incubation
with RA for an additional week, the spheres displayed
an increasing amount of bIII-tubulin-stained cells (Fig. 2d).
The immunoreactivity was not only expressed in the cell
bodies but also in the emerging neuronal processes (Fig. 2d
and f). We counted about 46% bIII-tubulin- and 63%
nestin-positive cells (Fig. 2g and h). An increase in the
level of bIII-tubulin, elaboration of neuronal processes
together with reduction in the level of nestin upon longer
incubation with RA indicated the progress of neuronal
differentiation.
(a) (b) (c)
(d) (e)
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841
(g)
(i)
NO signaling regulates neuronal precursor motility | 1831
(h)
Studies on gene expression of NT2 cell during and
shortly after differentiation with RA showed down-regulations
of several stem cell markers and up-regulation of
nestin and neurofilament transcripts that support our present
observations at the protein levels (Houldsworth et al. 2002;
Przyborski et al. 2003). Because of their high cellular
motility we further characterized the type of cells that
migrated out of 1-week-old NT2 spheres. Only 3.5 ± 0.5%
of the migratory cells were bIII-tubulin-positive and almost
all of the furthest migrated cells lacked the bIII-tubulin
staining (Fig. 2c). Hence, the cells at the front of the
migration expressed mainly the precursor marker nestin.
Costaining of BrdU together with nestin indicated that most
of the proliferating cells were nestin-positive neuronal
(f)

1832 | M. A. Tegenge and G. Bicker
(a) (b)
(c) (d)
(e) (f)
(g) (h)
progenitor cells (Fig. S1a). Costaining of BrdU together
with bIII-tubulin showed that none of the BrdU-incorporated
cells were bIII-tubulin positive (Fig. S1b). Thus,
Fig. 2 NT2 cell spheres express both nestin
and bIII-tubulin. NT2 cell spheres were
treated with 10 lM RA for a period of 1
and 2 weeks with medium change every
2–3 days. At the end of a 48 h migration
experiment, spheres were fixed and stained
against nestin and bIII-tubulin. One-weekold
NT2 spheres (a) express higher level of
nestin (green) than 2-week-old spheres (b).
The expression of bIII-tubulin in cell soma
and neuronal processes (red) after 2 weeks
RA treatment (d) increases in comparison
with the 1-week-old counterparts (c). Panels
(e–h) were obtained from spheres that
were mechanically dispersed into single
cells. Quantification of positively stained
cells showed that upon RA treatment for
2 weeks the level of nestin (g) was reduced
while bIII-tubulin (h) was significantly
increased. Blue (4¢-6-diamidino-2-phenylindole)
indicates nuclear counter-staining.
Scale bars: 100 lm (*p < 0.05, ***p <
0.001).
migration as measured in our cell culture model was mainly
based on the motility of nestin-positive neural precursor
cells (Fig. 2a). Along these lines we used the NT2 cell
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841

spheres to study the role of NO signaling in neuronal
precursor migration.
Agents that modulate cell migration via NO signaling do
not alter cell viability and differentiation
We initially examined the potential cytotoxicity of bioactive
chemical compounds affecting cell migration via NO/cGMP
signal transduction (Fig. 3a). Thus, we performed Alamar
Blue cell viability and the live/dead assay under identical
Fig. 3 Schematic drawing of NO signal
transduction together with an array of
chemical compounds that block or activate
the pathway (a). The enzyme nitric oxide
synthase (nNOS) is stimulated by calcium–
calmodulin (Ca/CaM) and can be blocked
by bath application of the inhibitor, 7-nitroindazole
(7NI). NO binds to the heme moiety
of soluble guanylyl cyclase (sGC)
resulting in the stimulation of the enzyme.
The sGC activity is blocked by the inhibitor
ODQ and stimulated independently from
NO by the sGC activator YC-1. Synthesis of
cGMP activates protein kinase G (PKG)
and regulates cell migration. The PKG
inhibitor, RP-8-Br-cGMP, blocks cellular
responses of the cGMP/PKG pathway. The
NO donors (SNP and NOC-18) and the
membrane-permeable cGMP analog (8-BrcGMP)
can be applied to raise cGMP
levels. The Live/Dead and Alamar Blue
viability/cytotoxicity assay was performed in
the presence of chemical compounds that
modulate the NO/cGMP signaling. Application
of 7NI (500 lM), ODQ (50 lM), RP-8-
Br-cGMP (100 nM), NOC-18 (50 lM), and
8-Br-cGMP (1 mM) to NT2 spheres did not
affect cell viability as determined by Live/
Dead assay (b) and Alamar Blue assay (c).
Relatively higher concentrations (1 mM) of
7NI, NOC-18, and ODQ significantly affected
cell viability as determined by Alamar
Blue assay (d–f). Application of 7NI
(500 lM), ODQ (50 lM), RP-8-Br-cGMP
(100 nM), NOC-18 (50 lM), and 8-BrcGMP
(1 mM) to NT2 spheres did not significantly
alter the bIII-tubulin-stained cells
(g). NOC-18, 2, 2-hydroxynitrosohydrazinobis-ethanamine;
RP-8-Br-cGMP, RP isomer
of 8-Br-cGMP; Ctrl, control (***p < 0.001).
(a)
experimental conditions as the migration assay. Both assay
showed that none of the pharmacological agents had any
effect on cell viability at the concentration used to modulate
cell migration (Fig. 3b and c). This was also further
confirmed by morphological observation of the spheres
including the migrating cells in the presence of the
compounds. However, when the NOS inhibitor, 7NI; sGC
inhibitor; ODQ; and NO donor, NOC-18 were used at the
rather high concentration of 1 mM, they significantly
(b) (c)
(d) (e)
(f) (g)
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Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841
NO signaling regulates neuronal precursor motility | 1833

1834 | M. A. Tegenge and G. Bicker
affected the survival of cells (Fig. 3d–f). Next, we used the
bIII-tubulin immunocytochemical staining to determine
the effect of chemicals on neuronal differentiation. At the
concentration used to modulate cell migration, only 7NI
appeared to slightly decrease while 8-Br-cGMP increased the
bIII-tubulin staining. However, the effect of both chemicals
was not statistically significant (Fig. 3g).
NT2 cells express nNOS and functional sGC
Western blotting revealed the presence of the nNOS in both
RA-treated and untreated precursor cells (Fig. 4a). Using a
(a)
NT2 + RA
NT2
155 kD
55 kD
(b) Ctrl (c)
(d) SNP + ODQ (e)
ODQ
monoclonal antibody against nNOS, the blot resolved a
major protein band at 155 kDa. This result is in line with
studies showing that untreated NT2 cells express nNOS but
not endothelial NOS and inducible NOS (Lee et al. 2001;
Hyun et al. 2002). For comparison of the NOS content in
precursor NT2 with NT2 + RA-treated cells, the ratio of
nNOS signal to the loading control was calculated and
averaged for all probed blots (n = 3). We found no
significant differences between NT2 + RA and NT2 cells
(0.76 ± 0.07 vs. 0.68 ± 0.11, respectively). To reveal
potential cellular targets of a NO signal endogenous to the
SNP
Fig. 4 NT2 cells express nNOS and functional
sGC. Western blotting of lysates from
NT2 cell spheres treated with RA for
1 week and untreated NT2 cells. The antibody
against nNOS recognizes a protein
band of apparent molecular weight at
155 kDa (a). Lower band represents a
loading control probed with acetylated-atubulin
antibody. Immuncytochemical
staining of cell spheres for cGMP levels (b–
e). After 48 h of seeding the spheres in
PDL- and Matrigel-coated cover glasses,
the cultures were exposed for 20 min to
SNP (1 mM), ODQ (50 lM), or a combination
of SNP and ODQ together with 1 mM
3-isobutyl-1-methylxanthine (IBMX) and
20 lM YC-1. Controls were also treated
with IBMX and YC-1. Cultures were fixed
with 4% PFA and stained against cGMP.
NT2 cell sphere shows very low cGMP-IR
(b). The addition of NO donor SNP increases
the cGMP-IR (c). The use of SNP
together with ODQ resulted in reduced
cGMP-IR (d). The cGMP-IR is almost
abolished in the presence of sGC inhibitor,
ODQ (e). Scale bar: 100 lm. Ctrl, control.
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841

NT2 cell spheres, we used an antibody against cGMP (de
Vente et al. 1987; Tanaka et al. 1997). In the absence of an
exogenous source of NO, a low level of cGMP was detected
which further confirmed the presence of an endogenous
source of NO (Fig. 4b). The level of cGMP increased
dramatically upon stimulation with the NO donor SNP
(Fig. 4c). We could not observe the increment in cGMP-IR
when SNP stimulation was accompanied by the sGC
inhibitor, ODQ (Fig. 4d). When ODQ was used alone,
hardly any cGMP-IR became detectable (Fig. 4e). All these
experiments showed the expression of a NO-sensitive sGC in
cells of the NT2 sphere culture.
Fig. 5 The migration of cells out of NT2
spheres was blocked in the presence of
NOS (7NI), sGC (ODQ), and PKG (RP-8-
Br-cGMP) inhibitors. Migration was determined
after 24 h of exposure to chemical
inhibitors. Representative photomicrographs
of NT2 spheres incubated as control
(Ctrl) only with RA and 0.25% dimethylsulfoxide
(DMSO) containing medium (a),
500 lM 7NI (b), and 50 lM ODQ (c). Dosedependent
inhibition of cell migration in the
presence of 7NI (d), ODQ (e), and RP-8-BrcGMP
(f). Scale bar: 100 lm. RP-8-BrcGMP,
RP isomer of 8-Br-cGMP (**p <
0.01, ***p < 0.001 compared to control.).
NO is a positive regulator of cell migration via the cGMP
and PKG pathway
As NO/cGMP signal transduction could be a positive
regulator of cell motility during neural development, we
examined the effect of nNOS inhibition on migratory
behavior of the differentiating NT2 cells. In the presence of
nNOS inhibitor, 7NI, the migration of cells out of the sphere
was significantly reduced (Fig. 5a, b and d). Application of
the sGC inhibitor, ODQ, at a concentration of 200 lM
decreased the migration of the cells by 60%. In a concentration
range of 50–200 lM, ODQ inhibited cell migration
in a dose-dependent manner (Fig. 5c and e). Potential
(a) (b)
(c) (d)
(e) (f)
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Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841
NO signaling regulates neuronal precursor motility | 1835

1836 | M. A. Tegenge and G. Bicker
downstream effector proteins for the cGMP signaling
pathway are PKG that exist in two isoforms, PKG-I and
PKG-II (Madhusoodanan and Murad 2007). The irreversibly
PKG-I binding inhibitor RP-8-Br-cGMP blocked the migration
of cells in a dose-dependent manner (Fig. 5f).
To test whether cell migration could be enhanced by an
exogenous stimulation of NO/cGMP signaling, we applied
the NO donor NOC-18 which decays with long half-life
(about 57 h at 22°C). After 24 h exposure to 50 lM of NOC-
18, the migration of cells increased by more than twofold.
Furthermore, NOC-18 significantly enhanced the migration
in a dose-dependent manner in the concentration range of 1–
100 lM (Fig. 6a–c). At relatively higher concentration
(1 mM), NOC-18 appeared to significantly inhibit cell
migration (Fig. 6c). A viability assay and morphological
observation showed that 1 mM of NOC-18 significantly
affected cell survival (Fig. 3e). The inhibitory effect on cell
migration at this high concentration seemed to be because of
cytotoxicity (Figs. 3e and 6c). As we determined cell
migration based on the furthest migrated cells, an increased
rate of cell proliferation might contribute to the facilitation of
migration. Hence, we conducted a BrdU cell proliferation
assay (Fig. 6d–h) in addition to viability testing. At the
concentration range where we found an increasing trend of
cell migration (1–50 lM), NOC-18 seemed to have a modest
but not significant effect on cell proliferation (Fig. 6d–g).
Intriguingly, 100 lM of NOC-18 appeared to facilitate cell
migration while inhibiting proliferation (Fig. 6c,f and g). A
BrdU proliferation assay was also performed in the presence
of nNOS/sGC/PKG inhibitors. We could not observe any
effect on proliferation of NT2 cell spheres at the concentration
of chemical inhibitors that blocked cell motility
(Fig. 6h). Additionally, migration was determined as the
distance of the furthest migrated nestin-positive cells after
NOC-18 treatment. We found out a significantly higher
migration distance of the nestin-positive cells after 50 lM
NOC-18 applications (Fig. 7a–c) suggesting that NOC-18
facilitated the migration of neuronal precursor cells. A BrdU
proliferation assay was also performed for the nestin-positive
cells which further confirmed that 50 lM NOC-18 did not
alter the neuronal precursor cell proliferation (Fig. 7d).
To demonstrate that the enhanced migration by exogenous
NO application was mediated via the cGMP pathway
(Fig. 3a), we exposed the NT2 cell spheres to NOC-18 while
blocking the downstream target enzymes sGC and PKG. We
observed a significant reduction of NO-induced facilitation of
cell migration when NOC-18 was used in combination with
ODQ or RP-8-Br-cGMP (Fig. 8a). This provided direct
evidence for the involvement of cGMP and PKG in NOmediated
neuronal precursor cell migration. Furthermore, a
direct facilitation of cell migration was observed by a cell
permeable analog of cGMP (8-Br-cGMP) (Fig. 8b–d). A
rescue experiment showed that 8-Br-cGMP reversed the
inhibitory effect of ODQ to the control level (Fig. 8e).
Discussion
NT2 cell spheres as a model for early developing human
neuron
The Ntera2 clone, D1 (NT2), is a well characterized cell line
that has been derived from a human testicular cancer. NT2
cells terminally differentiate into post-mitotic neurons by
exposure to micromolar doses of RA and mitotic inhibitors
(Andrews 1984; Pleasure et al. 1992; Paquet-Durand et al.
2003). The differentiated neurons express a large number of
neuronal markers and form functional synapses [for review
see Paquet-Durand and Bicker (2007)].
Neurosphere-based cultures have been employed to study
the involvement of signaling pathways that regulate neuronal
precursor cell proliferation, differentiation, and migration
(Leone et al. 2005; Mizuno et al. 2005; Moors et al. 2007).
Here, we used 10 lM of RA to generate NT2 aggregate
cultures that expresses both neuronal progenitor and early
neuronal markers. When the cultures were seeded on PDL
and Matrigel-coated substrate, cells could migrate out of the
spheres. Most of the cells that migrated out after 1 week RA
treatment were nestin-positive with only few cells displaying
bIII-tubulin staining. This indicates that the migrating cell
population is mainly composed of neuronal precursor cells
(Fig. 2a and c). When the time of RA application was
prolonged to 2 week, we observed an increase in the level of
bIII-tubulin in cell somata and the neuronal processes.
Expression of neuronal markers such as glutamate receptors,
MAP2, Tau, and NeuN just after 14-day exposure with RA
has been reported previously for NT2 cells (Megiorni et al.
2005). Furthermore, the migration assay showed that upon
prolonged treatment with RA, the cells tended to remain
inside the sphere and send out neuronal processes. This
indicates that RA treatment induces neuronal differentiation
while decreasing cell migration (Figs. 1 and 2). RA has been
shown to reduce the migration of airway smooth muscle cells
(Day et al. 2006) and neuroblastoma cell lines (Voigt and
Zintl 2003; Messi et al. 2008). The early expression of
cytoskeletal neuronal markers that resemble mammalian
neurogenesis (Przyborski et al. 2000, 2003; Houldsworth
et al. 2002; Paquet-Durand and Bicker 2007) suggests that
the NT2 aggregate culture system is a valid model for
developing human neuronal cells.
NO/cGMP/PKG pathway mediates the migration of human
neuronal precursor cells
In this study we showed for the first time that NO signaling
positively regulated cell migration in a model of human
neuronal precursor cells. Both undifferentiated NT2 precursors
and differentiating NT2 cell spheres expressed nNOS
(Fig. 4a) that could serve as endogenous source of NO. The
presence of functional sGC was directly demonstrated by
exogenous stimulation of cells with a NO donor and
subsequent immunocytochemical detection of cGMP. The
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841

(a) (b) (c)
(d) (e)
(g) (h)
Fig. 6 The NO donor (NOC-18) facilitates cell migration in a dosedependent
manner. Migration distance was determined after 24 h of
exposure to NOC-18. Representative photomicrographs of NT2 cell
spheres under control condition (a) and exposed to 50 lM of NOC-18
(b). Significant increase in cell migration in the presence of NOC-18 at
a concentration range of 1–100 lM (c). BrdU immunofluorescence
(intensity inverted) in a control (d), 50 lM (e), and 100 lM (f) of NOC-
18. 4¢-6-diamidino-2-phenylindole (DAPI) staining is shown at the
NO-induced cGMP-IR was reduced by the specific sGC
inhibitor ODQ (Fig. 4b–e). After showing the presence of the
NO signal transduction pathway in the NT2 cultures we
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841
NO signaling regulates neuronal precursor motility | 1837
(f)
bottom of panels (d–f). NOC-18 did not affect cell proliferation in a
concentration range of 1–50 lM; however, at 100 and 1000 lM, it
significantly inhibits cell proliferation (g). Application of 7NI (500 lM),
ODQ (50 lM), and RP-8-Br-cGMP (100 nM) to NT2 cell spheres did
not alter cell proliferation (h). Scale bars: 100 lm. NOC-18, 2, 2-hydroxynitrosohydrazino-bis-ethanamine;
RP-8-Br-cGMP, RP isomer of
8-Br-cGMP; Ctrl, control (*p < 0.05, **p < 0.01, ***p < 0.001 compared
to control.).
asked whether NO modulated the motility of cells out of the
spheres. Chemical manipulation of the target enzymes
involved in NO transduction demonstrated that inhibition

1838 | M. A. Tegenge and G. Bicker
(c)
(d)
(a) (b)
Fig. 7 NOC-18 facilitates the migration of nestin-positive precursor
cells. Migration was determined after 48 h of exposure to NOC-18,
fixed and stained for nestin. Representative photomicrographs of NT2
cell spheres under control condition (a) and exposed to 50 lM of
NOC-18 (b). The white lines in a and b delineate distance of migration
from the rim of the sphere to the furthest migrated nestin-positive cells.
Fifty lM of NOC-18 significantly increased the migration of the nestinpositive
neuronal precursor cells (c) and the % BrdU staining of the
nestin-positive cells was not altered by NOC-18 application (d). NOC-
18, 2, 2-hydroxynitrosohydrazino-bis-ethanamine; Ctrl, control (***p <
0.001).
of nNOS, sGC, and PKG reduced the motility of cells
(Fig. 5a–f). These loss of function effects suggested that NO
signal transduction positively regulated cell motility. Possible
cytotoxic effects of the inhibitors could be excluded as the
concentrations we used were well below the dose that caused
significant cell death (Fig. 3b–f).
A second line of evidence derives from a gain of function
after the exogenous application of NO and cGMP. The NO
donor and membrane permeable analog of cGMP enhanced
cell migration (Figs. 6a–c,7a–c and 8b–d) which directly
confirmed the positive regulatory role of NO and cGMP.
Interestingly, NO-induced facilitation of cell migration was
abolished when NOC-18 was used together with the sGC
inhibitor ODQ or PKG inhibitor RP-8-Br-cGMP (Fig. 8a).
This inhibition of migration by sGC and PKG inhibitors
infers that a certain level of cGMP is required for cell
motility. As pharmacological inhibition of the sGC enzyme
can be completely rescued by application of the cGMP
analog (Fig. 8e), it is unlikely that unspecific side effects of
the chemical blocker contribute to inhibition of cell migration.
Our combined result implicate that NO facilitates cell
migration by inducing sGC to increase cGMP levels which in
turn may activate PKG.
NO signal transduction and neuronal motility
Nitric oxide and cGMP have been shown to mediate the
migration of various cell types including neutrophils,
epithelial, and endothelial cells (Ziche et al. 1994; Elferink
and VanUffelen 1996; Noiri et al. 1996, 1998). Chemical
manipulations of cultured nervous systems implicate NO/
cGMP as regulator also for neuronal cell motility [reviewed
by Bicker (2005, 2007)]. Neuroanatomical studies using
markers against NOS and sGC suggest the migrating
neuroblasts of the rostral migratory stream as potential
targets for NO signaling in the adult brain (Moreno-López
et al. 2000; Gutièrrez-Mecinas et al. 2007). NO signal
transduction pathways regulate the migration of cerebellar
neurons, insect enteric neurons, and early developing
Xenopus neuronal cells (Tanaka et al. 1994; Haase and
Bicker 2003; Peunova et al. 2007; Knipp and Bicker 2009).
The application of a NO donor to young adult rats before
and after stroke increased cell proliferation and migration in
the SVZ and dentate gyrus (Zhang et al. 2001). Some
studies report that NO has no effect on post-mitotic
neuronal cell migration (Moreno-López et al. 2004) or
decreased ependymal/SVZ cell migration after focal cerebral
ischemia in rats (Zhang et al. 2007). Such inconsistency
in the effects of NO on neuronal migration could be
because of the use of different drug concentrations, species
differences, pathological, and developmental stages of the
experimental animals. Here, we provide further evidence for
a significant role of NO in human neuronal precursor cell
migration. The present study shows a concentration-dependent
facilitation of human neuronal precursor cell migration
by NO donor application. The inverted U-shaped dose-–
response curve (Fig. 6c) might in part account for the
inconsistency in the role of NO. In our cell culture model,
NOC-18 increased the migration of cells out of spheres in a
concentration range of 1–50 lM without affecting cell
viability and proliferation (Figs. 3e and 6a–g). Analysis of
migration distance and proliferation of the nestin-positive
cells confirmed that NOC-18 facilitated the migration of the
neuronal precursor cells (Fig. 7a–c) without affecting
proliferation (Fig. 7d). At 100 lM NOC-18 cell migration
was enhanced while proliferation was reduced (Fig. 6c, f
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841

Fig. 8 NO facilitates migration of cells out
of spheres via the cGMP and PKG pathway.
NO-induced facilitation of migration
was blocked in the presence of sGC (50 lM
ODQ) and PKG (100 nM RP-8-Br-cGMP)
inhibitors (a). Direct facilitation of cell
migration by cGMP analog (8-Br-cGMP) (b–
d). Inhibitory effect of ODQ (50 lM) was
rescued to the control level with 8-Br-cGMP
(1 mM) (e). RP-8-Br-cGMP, RP isomer of
8-Br-cGMP; Ctrl, control (**p < 0.01, ***p <
0.001 compared to control.)
and g). This result implicates that NO differentially
coordinates cell migration and proliferation, as has also
been reported by Peunova et al. (2007). At the relatively
high concentration of 1 mM, the increment in cell migration
was not observed. This reduction was most probably
because of cytotoxicity (Figs. 3e and 6g). Low concentration
of exogenous NO has been shown to enhance random
migration of neutrophils by stimulating sGC, while a higher
concentration inhibited migration (Elferink and VanUffelen
1996). Presumably, at low concentration NO activates only
its main target enzyme sGC (Garthwaite 2008) whereas at
sufficiently higher concentrations it rapidly reacts with
proteins and may even cause cell death [for review see
(a)
(b) (c)
(d) (e)
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841
NO signaling regulates neuronal precursor motility | 1839
Blaise et al. (2005) and Madhusoodanan and Murad
(2007)].
Presently, we do not know how the NO/cGMP/PKG
pathway causes the cytoskeletal reorganization that leads to
cell migration out of the NT2 spheres. In primary cultured
smooth muscle cells, the migration stimulatory effect of
NO donors and cGMP is associated with altered cell
morphology and dissociation of actin filaments (Brown
et al. 1999). In insect enteric neurons, where the NO/
cGMP/PKG pathway is crucial for cell migration, a block
of this pathway results in a relocalization of F-actin
bundles from the neurites to the cell body (Haase and
Bicker 2003). The stimulation of the NO/cGMP/PKG

1840 | M. A. Tegenge and G. Bicker
pathway in astrocytes caused a redistributation of glial
fibrillary acidic protein and depolymerization of actin with
a loss of stress fibers. These cytoskeletal rearrangements
resulted in a facilitation of astrocyte motility (Borán and
García 2007). Such changes in neurofilaments and actin are
attributed to inhibition of RhoA GTPase by NO-stimulated
PKG pathway (Sawada et al. 2001; Gudi et al. 2002; Borán
and García 2007; Peunova et al. 2007). Thus, there is a
link between the NO signal transduction pathway and actin
cytoskeleton, possibly via RhoA GTPase. Phosphorylation
of Enabled/vasodilator-stimulated phosphoprotein family
proteins that regulate actin polymerization by PKG has
also been reported to account for the action of cGMP in
cell motility (Sporbert et al. 1999; Chen et al. 2007;
Lindsay et al. 2007). Most likely changes in the cytosolic
calcium concentration will contribute to the morphology of
motile cell processes. Recent investigations have shown
that NO regulates growth cone filopodial behavior involving
sGC, PKG, and cyclic adenosine diphosphate ribose which
causes the release of calcium from intracellular stores via
the ryanodine receptor (Welshhans and Rehder 2005, 2007).
This study demonstrated that NO facilitated the migration
of differentiating model neuronal cells through the cGMP
and PKG signaling pathway. In our accessible NT2 sphere
culture system we can now investigate the mechanisms by
which NO regulates human neuronal precursor motility.
Acknowledgements
We thank Dr. J. de Vente for his kind gift of the cGMP antiserum,
Dr M. Stern for the discussion and help during the preparation of the
manuscript, and S. Tan for technical support. M.A. Tegenge was
supported by a Georg–Christoph–Lichtenberg scholarship from the
Ministry for Science and Culture of Lower Saxony and G. Bicker
was supported by DFG grant BI 262/16-1.
Supporting Information
Additional Supporting Information may be found in the online
version of this article:
Figure S1. BrdU is incorporated into the nestin-positive neuronal
precursor cells.
As a service to our authors and readers, this journal provides
supporting information supplied by the authors. Such materials are
peer-reviewed and may be re-organized for online delivery, but are
not copy-edited or typeset. Technical support issues arising from
supporting information (other than missing files) should be
addressed to the authors.
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JOURNAL OF NEUROCHEMISTRY | 2009 | 111 | 1434–1446 doi: 10.1111/j.1471-4159.2009.06421.x
, ,
*Division of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, Hannover, Germany
Center for Systems Neuroscience (ZSN), Hannover, Germany
Abstract
The human Ntera2 (NT2) teratocarcinoma cell line can be
induced to differentiate into post-mitotic neurons. Here, we
report that the human NT2 neurons generated by a spherical
aggregate cell culture method express increasing levels of
typical pre-synaptic proteins (synapsin and synaptotagmin I)
along the neurite depending on the length of in vitro culture.
By employing an antibody directed against the luminal domain
of synaptotagmin I and the fluorescent dye N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridiniumdibromide,
we show that depolarized NT2 neurons display
calcium-dependent exo-endocytotic synaptic vesicle recycling.
NT2 neurons express the neuronal isoform of neuronal
nitric oxide synthase and soluble guanylyl cyclase (sGC), the
major receptor for nitric oxide (NO). We tested whether NO
The gaseous messenger nitric oxide (NO) which is produced
by the enzyme nitric oxide synthase has been extensively
studied as a retrograde messenger that modulates synaptic
plasticity and memory formation (Boehning and Snyder
2003; Garthwaite 2008; Taqatqeh et al. 2009). The major
physiological target of NO is the cylic guanosine-monophosphate
(cGMP)-synthesizing enzyme soluble guanylyl
cyclase (sGC). Numerous studies indicate that NO can
induce transmitter release at pre-synaptic terminals (Hawkins
et al. 1994; Arancio et al. 1996; Meffert et al. 1996; Sporns
and Jenkinson 1997; Wildemann and Bicker 1999; Li et al.
2002; Nickels et al. 2007).
Mechanisms by which NO induces vesicle release are
gradually being discovered. Enhancement of transmitter
release via the cGMP pathway which certainly involves
phosphorylation via protein kinase G (PKG) has been
suggested as a major mechanism (Arancio et al. 1995,
2001; Lu et al. 1999; Wildemann and Bicker 1999; Li et al.
2004; Garthwaite 2008). Moreover, post-translational modification
of ion channels and proteins involved in vesicle
docking/fusion processes by S-nitrosylation has also been
signal transduction modulates synaptic vesicle turnover in
human NT2 neurons. NO donors and cylic guanosine-monophosphate
analogs enhanced synaptic vesicle recycling while
a sGC inhibitor blocked the effect of NO donors. Two NO
donors, sodium nitroprusside, and and N-Ethyl-2-(1-ethyl-2hydroxy-2-nitrosohydrazino)
ethanamine evoked vesicle exocytosis
which was partially blocked by the sGC inhibitor. The
activator of adenylyl cyclase, forskolin, and a cAMP analog
induced synaptic vesicle recycling and exocytosis via a parallel
acting protein kinase A pathway. Our data from NT2
neurons suggest that NO/cyclic nucleotide signaling pathways
may facilitate neurotransmitter release in human brain cells.
Keywords: cyclic AMP, cyclic GMP, exocytosis, FM1-43,
nitric oxide, NTera2.
J. Neurochem. (2009) 111, 1434–1446.
reported (Meffert et al. 1996; Ahern et al. 2002). The NO/
cGMP pathway has been shown to regulate synaptic vesicle
endocytosis and recycling by increasing pre-synaptic
Received August 7, 2009; revised manuscript received September 16,
2009; accepted September 16, 2009.
Address correspondence and reprint requests to Gerd Bicker, Division
of Cell Biology, Institute of Physiology, University of Veterinary Medicine
Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany.
E-mail: gerd.bicker@tiho-hannover.de
Abbrevations used: 8-Br-cAMP, 8-Bromoadenosine 3¢, 5¢-cyclic
adenosine monophosphate, sodium salt; cGMP, cylic guanosine-monophosphate;
cGMP-IR, cGMP-immunoreactive; DIV, days in vitro; FM1-
43, N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium
dibromide; FM1-43Fx, fixable analog of FM1-43; H-89, N-[2-((p-
Bromocinnamyl) amino) ethyl]-5-isoquinolinesulfonamide, 2HCl; KRH,
Krebs-Ringer’s – HEPES; nNOS, neuronal nitric oxide synthase; NO,
nitric oxide; NOC-12, N-Ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)
ethanamine; NT2, Ntera2; ODQ, 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one;
PBS, phosphate-buffered saline; PFA, 4%
paraformaldehyde in PBS; PKA, protein kinase A;PKG, protein kinase
G;Rp-cAMP, adenosine 3¢,5¢-cyclic monophosphorothioate, Rp-isomer;sGC,
soluble guanylyl cyclase;SNP, sodium nitroprusside.
Ó 2009 The Authors
1434 Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 1434–1446

phosphatidylinositol 4, 5-bisphosphate (Micheva et al.
2003). In addition to its role as a retrograde messenger,
NO has been implicated to play a critical role in early
neuronal development including cell proliferation, migration,
and synaptogenesis (Enikolopov et al. 1999; Bicker 2005,
2007). However, the limited availability of in vitro models
for the developing human brain has restricted the analysis of
potential roles for NO signals in vesicle turnover, a pathway
that may also contribute to activity-dependent nerve cell
development.
In this study, we used the well-characterized human
teratocarcinoma cell line Ntera2 (NT2) that can be induced to
differentiate into neurons upon treatment with retinoic acid as
surface-attached adherent monolayer culture (Andrews 1984;
Pleasure et al. 1992). In recent years, this rather lengthy
differentiation method was significantly reduced by employing
a cell aggregate culture method (Paquet-Durand et al.
2003; Podrygajlo et al. 2009). Despite their common clonal
origin, differentiated NT2 neurons comprise a heterogeneous
population of cells expressing several neurotransmitters in
vitro (Guillemain et al. 2000; Podrygajlo et al. 2009).
Electron microscopical and immunocytochemical studies
revealed the presence of pre-synaptic vesicles and synaptic
vesicle-associated proteins such as synaptobrevin, synaptophysin,
and synapsin (Sheridan and Maltese 1998; Hartley
et al. 1999; Guillemain et al. 2000; Podrygajlo et al. 2009).
NT2 neurons also express voltage-gated Na channels
(Matsuoka et al. 1997) and L, N, P/Q and R-type Ca
channels that are recruited for neurotransmission (Gao et al.
1998; Neelands et al. 2000).
Here, we characterized in aggregate culture generated
human NT2 neurons by monitoring luminal synaptotagmin I
immunoreactivity and imaging of synaptic vesicle recycling
with the fluorescent dye N-(3-triethylammoniumpropyl)-4-
(4-(dibutylamino)styryl)pyridinium dibromide (FM1-43).
Both methods revealed that depolarized NT2 neurons display
calcium-dependent synaptic vesicle recycling. By employing
immunocytochemical methods, we showed the expression of
neuronal nitric oxide synthase (nNOS) and functional sGC in
subpopulations of NT2 neurons. Finally, we used the human
NT2 neurons as a model to demonstrate the involvement of
NO/cGMP and cAMP/protein kinase A (PKA) signal
transduction in vesicle recycling and pre-synaptic vesicle
exocytosis.
Materials and methods
The NO donor N-Ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)
ethanamine (NOC-12), the PKA antagonists N-[2-((p-Bromocinnamyl)
amino) ethyl]-5-isoquinolinesulfonamide, 2HCl (H-89) and
adenosine 3¢, 5¢-cyclic monophosphorothioate, Rp-isomer, triethylammonium
salt were purchased from Calbiochem (Darmstadt,
Germany). 8-bromoguanosine-3¢, 5¢-cyclic monophosphate, sodium
salt and 8-bromoadenosine 3¢, 5¢-cyclic monophosphate, sodium
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 1434–1446
NO and cyclic nucleotide modulates vesicle release | 1435
salt (8-Br-cAMP) were purchased from Alexis Biochemicals
(Lörrach, Germany). All other materials were obtained from
Sigma (Taufkirchen, Germany) unless otherwise noted. Buffers
with the following compositions were prepared: phosphate-buffered
saline (PBS; 10 mM sodium phosphate, 150 mM NaCl, pH
7.4), Krebs-Ringer’s – HEPES (KRH) (in mM; 115 NaCl, 5 KCl,
1 MgCl 2 · 6H 2O, 24 NaHC0 3, 2.5 CaCl 2.2H 2O, 25 Glucose, 25
HEPES, pH 7.4), calcium free KRH that contains 2.5 mM EGTA
instead of CaCl 2 · 2H 2O (Ca 2+ free), KRH buffer that contains
60 mM of KCl, corrected for osmolarity by reduction of NaCl
(high K + ) and high K + without CaCl 2 · 2H 2O (Ca 2+ free high K + ).
All chemicals were diluted in KRH at the following final
concentrations: sodium nitroprusside (SNP, 1 mM), NOC-12
(100 lM), 1H-[1, 2, 4]-oxadiazolo [4, 3-a] quinoxalin-1-one
(ODQ, 50 lM), forskolin (50–100 lM), 8-bromoguanosine-3¢, 5¢cyclic
monophosphate, sodium salt (100–1000 lM), 8-Br-cAMP
(100–1000 lM), adenosine 3¢, 5¢-cyclic monophosphorothioate,
Rp-isomer (Rp-cAMP) (10 lM) and H-89 (10 lM). 3-Isobutyl-1methylxanthine,
ODQ, and forskolin were prepared from stocks in
dimethylsulfoxide, which were further diluted in KRH to result in
a maximum concentration of 0.25% dimethylsulfoxide. Pathway
inhibitors were pre-incubated for 30 min with the NT2 neurons
prior to the experiments while activators were applied for 5 min
during loading of FM1-43.
Cell culture
The human NT2/D1 cell line was obtained from American Type
Culture Collection, Manassas, VA, USA. NT2 precursor cells were
maintained and cultivated in Dulbecco’s modified Eagle medium/
F12 (Gibco-Invitrogen, Karlsruhe, Germany) supplemented with
10% fetal bovine serum (Gibco-Invitrogen) and 1% penicillin/
streptomycin (Gibco-Invitrogen) in an atmosphere of 5% CO 2 at
37°C (Andrews 1984). Post-mitotic NT2 neurons were obtained by
free-floating spherical aggregate methods (Paquet-Durand et al.
2003; Podrygajlo et al. 2009). Briefly, NT2 precursor cells were
seeded in 95 mm bacteriological grade Petri dishes (Greiner,
Hamburg, Germany) for 24 h. Dulbecco’s modified Eagle medium/
F12 medium containing 10% fetal bovine serum and 10 lM retinoic
acid was used up to 1 week. The aggregates were mechanically
dispersed and treated in RA (retinoic acid) for one additional week in
T75 flasks as adherent culture. Finally, cells were trypsinized and
seeded in T75 flasks and supplied with culture medium containing
mitotic inhibitors (1 lM 1-6-D-arabinofuransylcytosine, 10 lM 2¢deoxy-5-fluorouridine,
10 lM 1-b-D-ribofuranosyluracil). After 7–
10 days, fully differentiated neurons were selectively trypsinized and
were plated on poly-D-lysine and Matrigel (Becton-Dickinson,
Bedford, MA, USA) coated 12 or 25 mm cover glasses at a density
of 20 000 or 100 000 cells/cover glass for further experimentation.
Immunofluorescence
Ntera2 neurons were washed with PBS and fixed for 30 min at
20°C with 4% paraformaldehyde in PBS (PFA). Immunofluorescence
detection of microtubule associated protein 2 (MAP2)
(1 : 1000; Sigma), Tau1 (1 : 500; Millipore International),
synapsin (1 : 500; Synaptic Systems, Göttingen, Germany), synaptotagmin
I directed to cytoplasmic domain (1 : 10, mAb30;
Developmental Studies Hybridoma Bank, Iowa, USA), and nNOS
(1 : 200; Sigma) was performed as previously described (Tegenge

1436 | M. A. Tegenge et al.
and Bicker 2009). For the detection of cGMP-immunoreactive
(cGMP-IR) cells, NT2 neurons were pre-incubated for 20 min at
20°C with 1 mM SNP as an NO donor, 20 lM 3-(50-Hydroxymethyl-20-furyl)-1-benzyl
indazole as an enhancer of NO-induced
activity of sGC, and 1 mM 3-isobutyl-1-methylxanthine as a
phosphodiesterase inhibitor in PBS. Cultures were washed once
with PBS and fixed with 4% PFA for 30 min. The polyclonal
sheep cGMP antiserum (1 : 10 000; a kind gift from Dr. J. de
Vente, Maastricht University, the Netherlands) was used as primary
antibody to detect the level of cGMP (Tegenge and Bicker 2009).
Luminal synaptotagmin I immunostaining
Ntera2 neurons were assayed for synaptic vesicle recycling using
polyclonal antibody directed against the luminal domain of
synaptotagmin I (Synaptic Systems). Cultures were incubated for
20 min with the antibody diluted 1 : 100 in high K + . For
comparison, incorporation of anti-luminal synaptotagmin I was
performed in normal KRH (basal), Ca 2+ free high K + , and SNP or
SNP + ODQ diluted in KRH. Cells were then fixed with 4% PFA
and the incorporated antibody was detected by immunofluorescence.
Western blotting
The protein content of cell lysate was estimated by the BCA TM
protein assay kit (Pierce, Rockford, IL, USA). Equal amount of
protein (50 lg) from NT2 cells and NT2 neurons was used. The
monoclonal anti-nNOS (Sigma) at 1 : 1000 dilution and antiacetylated-a-tubulin
(Sigma) diluted 1 : 10 000 were used for
western blotting as previously described (Tegenge and Bicker 2009).
FM1-43 imaging
The uptake and release of FM1-43 dye was performed as described
by Gaffield and Betz (2006). Briefly, for uptake experiments, NT2
neurons cultured for 3–5 weeks were washed with PBS and
stimulated for 5 min with high K + buffer in the presence of
10 lM fixable analog of FM1-43 (FM1-43Fx; Molecular Probes,
Eugene, OR, USA) and compared with the uptake of FM1-43 in
normal KRH (basal), Ca 2+ free high K + and chemicals. After FM1-
43Fx was loaded into pre-synaptic terminals, NT2 neurons were
washed twice in KRH followed by additional twice washing in Ca 2+
free buffer. Neurons were then fixed with 4% PFA and images were
taken immediately. The release of normal FM1-43 was followed in
real time after loading the NT2 neurons with 10 lM FM1-43
(Molecular Probes) in high K + buffer for 5 min. The culture was
transferred into a perfusion chamber, mounted on a Zeiss Axiovert
200 microscope and maintained at 37°C. Cultures were continuously
washed for 10 min in normal KRH in a perfusion chamber at
flow rate of 2–2.5 mL/min. Cultures were washed for additional
5 min in Ca 2+ free buffer. The unloading of FM1-43 was followed
upon stimulation of the culture with high K + ,Ca 2+ free high K + ,
normal KRH, or chemicals diluted in normal KRH.
Microscopy and data analysis
Preparations were viewed with a Zeiss Axiovert 200 (Göttingen,
Germany), equipped with a CoolSnap camera (Photometrics,
Tucson, AZ, USA) and MetaMorph software (Molecular Devices,
Sunnyvale, CA, USA). Confocal images of NT2 neurons loaded
with FM1-43Fx and co-stained against cytoplasmic domain of
synaptotagmin I were prepared using a Leica TCS-SP5 spectral laser
scanning confocal microscope (Leica Mikrosysteme Vertrieb
GmbH, Wetzlar, Germany) with LAS AF software and under 63·
oil immersion objective. For quantification of synapsin and
synaptotagmin levels, stained synaptic puncta were counted along
the length of a neurite and expressed as number of synaptic puncta
per number of neurons obtained from 4¢,6-diamidino-2¢-phenylindol-dihydrochloride
staining. To obtain an estimate of cGMP levels,
we counted cGMP-IR cell bodies. Data were presented as
mean ± SEM of at least eight fields of view (220 · 165 lm) from
at least three independent experiments. For FM1-43Fx uptake
experiments, fluorescence intensity of individual puncta (numbers
are given in the figure legends) was measured using Simple PCI
software (C-Imaging Systems, Sewickley, PA, USA). Background
fluorescence from a selected blank area was subtracted from
individual puncta. For FM1-43 unloading experiments, fluorescence
intensity was measured as above every 15 s for about 5 min. The
percent fluorescence intensity remaining at the end of stimulation
was calculated. Data were presented as mean ± SEM. Statistical
analysis was performed using unpaired Student’s t-test. Levels of
significance were: *p < 0.05, **p < 0.01, ***p < 0.001.
Results
NT2 neurons undergo pre-synaptic maturation
We used the aggregate culture method (Paquet-Durand
et al. 2003; Podrygajlo et al. 2009) to generate NT2
neurons expressing MAP2 and Tau, typical neuronal
cytoskeletal markers of dendrites and axons, respectively.
NT2 neurons cultured for 7 days in vitro (DIV) after
differentiation displayed MAP2 staining on short processes
originating from the cell somata (Fig. 1a). Only weak Taustaining
appeared on long-neuronal processes after 7 DIV
(Fig. 1b). NT2 neurons cultured for 28 DIV were stained
strongly for both MAP2 and Tau (Fig. 1e and f). To
monitor the progress of pre-synaptic maturation, fully
differentiated NT2 neurons were cultured in vitro for about
6 weeks and stained over time for the pre-synaptic proteins
synapsin and synaptotagmin I. After 7 DIV culture, NT2
neurons expressed very little punctate synapsin staining
that appeared mainly around cell somata (Fig. 1c). However,
within 14–28 DIV intense punctate synapsin staining
appeared on long neurites and around cell somata
(Fig. 1g). Similarly, only little punctate synaptotagmin I
staining was confined to the cell bodies of NT2 neurons
cultured for 7 DIV (Fig. 1d). Intense synaptotagmin I
staining appeared along the neurites within 14–28 DIV
cultures (Fig. 1h). We quantified the levels of synapsin and
synaptotagmin I as number of synaptic puncta per neuron.
The level of both proteins increased significantly within
14–28 DIV culture as compared with 7 DIV (Fig. 2a)
indicating that human NT2 neurons undergo pre-synaptic
maturation. In NT2 neurons cultured for about 6 weeks,
the level of synapsin and synaptotagmin did not increase
compared with 28 DIV old cultures (data not shown).
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 1434–1446

Fig. 1 Human NT2 neurons express typical
cytoskeletal neuronal marker and presynaptic
proteins. The maturation of NT2
neurons generated by aggregate culture
was followed by immunocytochemical
staining for (a, f) MAP2, (b, g) Tau, (c, h)
synapsin, (d, i) cytoplasmic, and (e, j)
luminal synaptotagmin I. After 7 days
in vitro (DIV), NT2 neurons were (a) intensely
stained for the neuronal marker
MAP2, (b) weakly stained for the axonal
marker, Tau. (c, d) Only little synapsin and
synaptotagmin puncta staining appeared on
NT2 neurons cultured for 7 DIV. After 28
DIV culture, NT2 neurons were intensely
stained for (f) MAP2 and (g) Tau. (h, i)
Intense synapsin and synaptotagmin staining
appeared along the neurites of NT2
neurons cultured for 28 DIV. (e, j) Presynaptic
terminals of NT2 neurons cultured
for 28 DIV were labeled with luminal
synaptotagmin I compared with 7 DIV culture.
Arrow heads (h, i and j) indicate
representative punctate staining along
the neurites. Blue (4¢,6-diamidino-2¢-phenylindol-dihydrochloride)
indicates nuclear
counter-staining. Scale bar: 200 lm (a, b,
f and g), 100 lm (c, d, h and i), 50 lm
(e and j). [Correction added after online
publication 9 November 2009; panel labelling
changed in figure legend].
(a) (f)
(b) (g)
(c) (h)
(d) (i)
(e) (j)
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 1434–1446
NO and cyclic nucleotide modulates vesicle release | 1437

1438 | M. A. Tegenge et al.
(a)
(b)
(c)
Fig. 2 Human NT2 neurons undergo pre-synaptic maturation. The
levels of synapsin and synaptotagmin I were quantified by counting the
number of synaptic puncta along the neurites in at least eight fields of
view taken from at least three independent experiments. (a) The
number of puncta staining for synapsin and synaptotagmin were significantly
increased within 14–28 DIV culture as compared with 7 DIV.
(b) Synaptic vesicle recycling was determined by antibody directed to
the luminal domain of synaptotagmin I during depolarization of human
NT2 neurons by high K + . The extent of synaptic vesicle recycling increased
significantly during the maturation of NT2 neurons within 14–
28 DIV. (c) The incorporation of luminal synaptotagmin I depends on
both depolarization and presence of calcium.
Depolarized human NT2 neurons display synaptic vesicle
recycling in a calcium-dependent manner
Antibodies directed to the luminal domain of synaptotagmin
I have been used to label pre-synaptic vesicles that undergo
exocytosis (Matteoli et al. 1992; Kraszewski et al. 1995;
Verderio et al. 2007). We applied the luminal synaptotagmin
I antibody in the presence of a depolarizing high K +
(60 mM) which causes multiple round of synaptic vesicle
recycling and hence incorporation of the antibody into the
nerve terminals. Upon stimulation with high K + , NT2
neurons (28 DIV) were intensely labeled with synaptotagmin
(Fig. 1j) indicating that the neurons perform synaptic vesicle
recycling. The immunoreactivity appeared as punctate staining
along the neurites and cell somata (Fig. 1j). We found on
average higher values of immunoreactive synaptic puncta in
NT2 neurons cultured for about 28 DIV than for 7 DIV
(Figs 1i, j and 2b). Thus, similar to the expression of the presynaptic
proteins, the activity-dependent immunostaining of
the neurons critically depended on the length of in vitro
culture. Quantification of the number of synaptic puncta
revealed that depolarization by high K + resulted in significantly
higher values of luminal synaptotagmin labeling
compared with the basal level (Fig. 2c). Application of antisynaptotagmin
antibody under high K + stimulation in the
absence of Ca 2+ resulted in significantly lowered numbers of
synaptic puncta (Fig. 2c). These results clearly demonstrate
that depolarized NT2 neurons show Ca 2+ -dependent synaptic
vesicle recycling.
To directly visualize synaptic exo- and compensatory
endocytosis, we used fluorescent FM-dyes that are trapped
in retrieved vesicle membranes in an activity-dependent
manner. Firstly, we performed vesicle uptake experiments
with the fixable dye FM1-43Fx as a monitor of synaptic vesicle
turnover (Fig. 3a–c). Upon stimulation with high K + , NT2
neurons were successfully loaded with FM1-43Fx as compared
with the basal level (Fig. 3a–c). Furthermore, the
loading of FM1-43Fx was significantly lowered when it was
applied with high K + in the absence of Ca 2+ (Fig. 3c). Then,
we imaged exocytosis by monitoring the dimming of fluorescence
when dye trapped in the synaptic vesicles is being
released. The unloading of FM1-43 was followed in real time
during second period of stimulation with high K + after dye
loading and washing. Upon stimulation, the NT2 neurons
released the dye within 5 min (Fig. 3d–g) indicating the presynaptic
vesicle exocytosis. The unloading of FM1-43 also
depended on the presence of calcium in the stimulation buffer
(Fig. 3g and h). To confirm that the FM1-43Fx loaded punctate
staining indicates pre-synaptic structure, we performed
co-staining of FM1-43Fx loaded neurons with the synaptotagmin
I antibody (Fig. 4a–c). Even though the staining for
FM1-43Fx weakened during permeabilization and washing of
the culture, we found that most of the FM-labeled puncta were
synaptotagmin I-immunoreactive (Fig. 4c).
NO and cyclic GMP modulates synaptic vesicle recycling
and exocytosis
The expression of nNOS by human NT2 neurons was
detected by western blotting which resolved a major protein
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 1434–1446

(a)
(d)
(g) (h)
Fig. 3 Human NT2 neurons display synaptic vesicle recycling and
exocytosis. NT2 neurons were cultured for 28 DIV and labeled with the
fixable analog of FM1-43 (FM1-43Fx) and normal FM1-43. (a, b) FM1-
43Fx is loaded into recycling synaptic vesicle of NT2 neurons upon
stimulation with high K + compared to basal (KRH). Photomicrographs
at the bottom of (a, b) represent the respective phase contrast images
of NT2 neurons. (c) Quantification of FM1-43Fx uptake as fluorescent
intensity of the basal level indicates significantly higher uptake of the
dye into NT2 neuron terminals upon stimulation by high K + as compared
with KRH and Ca 2+ free high K + . (d–h) The unloading of normal
FM1-43 from NT2 neurons was followed upon stimulation with high K +
band around 155 kDa (Fig. 5a). This is in line with previous
reports (Lee et al. 2001; Tegenge and Bicker 2009) for the
expression of nNOS in the NT2 precursor cells. Subpopulations
of NT2 neurons positively stained for the nNOS
monoclonal antibody (Fig. 5b). The immunofluorescence
labeling for nNOS appeared mainly around the cell somata
and weakly along the neurites of NT2 neurons (Fig. 5b). To
reveal the presence of the functional NO receptor sGC, we
used an antibody against cGMP (De Vente et al. 1987). In
(b)
(c)
(e) (f)
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 1434–1446
NO and cyclic nucleotide modulates vesicle release | 1439
with or without Ca 2+ . (d) FM1-43 loaded punctate staining after
washing with KRH (10 min) and calcium free buffer (5 min). (e, f) FM1-
43 loaded terminals undergo destaining within 5 min upon stimulation
with high K + . Arrow heads in (d–f) indicates representative puncta that
undergo destaining. (g) A representative FM1-43 destaining curve
upon stimulation by high K + with or without calcium. (h) The percent
fluorescence intensity remaining at the end of stimulation with high K +
was significantly lower than Ca 2+ free high K + . Data represent
mean ± SEM of at least 100 (c) and 20 (h) synaptic puncta from three
independent experiments. Scale bars: 50 lm.
the absence of an exogenous source of NO, on average
6.7 ± 1% cGMP-IR cells were detected (Fig. 5c). The
number of cGMP-IR cells increased significantly up to
49.4 ± 4% upon stimulation with the NO donor SNP
(Fig. 5d). The number of NO-induced cGMP-IR cells
reduced to 23.6 ± 4% when SNP stimulation was accompanied
by the sGC inhibitor, ODQ (Fig. 5e). Our data clearly
demonstrate the expression of a NO-sensitive sGC in
subpopulations of NT2 neurons.

1440 | M. A. Tegenge et al.
(a) (b) (c)
Fig. 4 FM1-43Fx loaded synaptic puncta co-localize with synaptotagmin I. NT2 neurons (21 DIV) were loaded with (a) FM1-43Fx, fixed and
stained against (b) cytoplasmic domain of synaptotagmin I. (c) FM1-43Fx loaded synaptic puncta were colocalized with synaptotagmin I. Scale bar:
25 lm.
(a)
(b)
(c) (d) (e)
Fig. 5 NT2 neurons express nNOS and functional sGC. (a) Western
blotting of cell lysate from NT2 precursor cells and NT2 neurons. The
antibody against nNOS recognizes a protein band of apparent
molecular weight at 155 kDa and the lower band represents acetylated
a-tubulin. (b) Immunofluorescence detection of nNOS in cell somata of
NT2 neurons and around the neurites. (c–e) cGMP-IR of NT2 neurons
for the detection of functional sGC. NT2 neurons cultured for 28 DIV
was exposed for 20 min to SNP (1 mM) or SNP + ODQ (50 lM) together
with 1 mM 3-isobutyl-1-methylxanthine (IBMX), as a phos-
To investigate the involvement of the NO-cGMP signal
transduction pathway in pre-synaptic neurotransmitter release,
we exposed NT2 neurons to the NO donor SNP. This
resulted in a significantly higher incorporation of the luminal
phodiesterase inhibitor and 20 lM of 3-(50-Hydroxymethyl-20-furyl)-1benzyl
indazole (YC-1), as an enhancer of NO-induced activity of sGC.
Controls were also treated with IBMX and YC-1. Cultures were fixed
with 4% paraformaldehyde in PBS and stained against cGMP. (c)
Under control conditions, NT2 neurons showed little cGMP-IR. (d) The
addition of the NO donor SNP increased the cGMP-IR. (e) Application
of SNP together with ODQ reduced NO-induced cGMP-IR. Blue in (c–
e) indicates nuclear counter-staining (4¢,6-diamidino-2¢-phenylindoldihydrochloride).
Scale bar: 50 lm (b), 100 lm (c–e).
synaptotagmin I antibody similar to depolarization-induced
labeling by high K + (Fig. 6a). The incorporation of antiluminal
synaptotagmin by SNP was blocked when it was
applied together with the sGC inhibitor, ODQ (Fig. 6a).
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 1434–1446

(a)
(b)
(c)
Fig. 6 NO and cGMP analog facilitate synaptic vesicle recycling. (a)
The NO donor, SNP (1 mM) significantly increased the incorporation
of luminal synaptotagmin I into NT2 neuron that was blocked by the
sGC inhibitor, ODQ (50 lM). (b) The uptake of FM1-43Fx was significantly
increased by SNP (1 mM). Pre-incubation with sGC inhibitor
(ODQ, 50 lM) blocked SNP-induced uptake of FM1-43Fx. (c) The cell
membrane-permeable analog of cGMP, 8-bromoguanosine-3¢, 5¢cyclic
monophosphate, sodium salt enhanced the uptake of FM1-
43Fx. Data represent mean ± SEM of percent fluorescence intensity
of at least 80 synaptic puncta from three independent experiments.
Likewise, the uptake of FM1-43Fx was enhanced by SNP
(Fig. 6b) and a membrane-permeable analog of cGMP
(Fig. 6c). Pre-incubation with ODQ resulted in blocking of
SNP-induced uptake of the dye (Fig. 6b). These experimental
results suggest that NO/cGMP modulates synaptic vesicle
recycling in NT2 neurons.
The unloading of FM1-43 was followed in real time by
monitoring the dimming of fluorescence intensity after
stimulation with two NO donors, SNP, and NOC-12. Both
NO donors induced the release of the dye with a destaining
profile of FM1-43 similar to that seen in high K + (Fig. 7a).
At the end of 5 min lasting stimulation with SNP or NOC-
12, the percentage of fluorescence remaining in NT2 nerve
terminals was significantly lower than in normal KRH,
indicating that NO-induced pre-synaptic vesicle exocytosis
(Fig. 7b). After FM1-43 was loaded into the neurons, the
culture was incubated with ODQ for 10 min before the
unloading of FM1-43 was initiated with SNP or NOC-12.
Pre-incubation with ODQ significantly blocked SNP
(Fig. 7b) and NOC-12 (Fig. 7c) induced FM1-43 unloading.
These results show that NO induces vesicle exocytosis in
NT2 neurons via the cGMP pathway.
cAMP/PKA signal transduction modulates synaptic vesicle
recycling and exocytosis
To reveal a possible involvement of the cAMP signaling
pathway, we used activators and inhibitors of this cascade in
FM1-43 uptake experiments. The uptake of FM1-43Fx was
significantly enhanced in the presence of the adenylyl
cyclase stimulator forskolin (Fig. 8a) and a cell membrane
permeable analog of cAMP (8-Br-cAMP) (Fig. 8b). Preincubation
of NT2 neurons with PKA inhibitors (Rp-cAMP
or H-89) significantly reduced forskolin-induced (Fig. 8c)
and high K + -induced (data not shown) uptake of FM1-43Fx
indicating that cAMP/PKA pathway positively regulates
synaptic vesicle recycling. Additionally, we followed the
release of FM1-43 upon stimulation with forskolin. Stimulation
of NT2 neurons with forskolin resulted in rapid
release of FM1-43 which indicates that forskolin induced
vesicle exocytosis (Fig. 8d). Pre-incubation with H-89
significantly reduced the release of FM1-43 indicating the
participation of PKA in forskolin induced vesicle exocystosis
(Fig. 8d).
Discussion
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 1434–1446
NO and cyclic nucleotide modulates vesicle release | 1441
Human NT2 neurons display synaptic vesicle recycling and
pre-synaptic release
Recently, we have shown that the human NT2 cells
proliferate during retinoic acid treatment as spherical
aggregate culture and cells migrate out of the aggregate
to acquire fully differentiated neuronal phenotypes (Tegenge
and Bicker 2009). In this study, we followed the progress of
NT2 neuronal maturation after the migratory phase when
neuronal networks are formed. After 7 DIV, intense MAP2
staining indicated full differentiation of NT2 neurons but
staining for phosphorylated Tau, indicative of axonogenesis,
was weak (Fig. 1a and b). However, after 28 DIV,
intense Tau-staining appeared on long neurites, whereas
MAP2 staining did not change in the cell cultures (Fig. 1e
and f).

1442 | M. A. Tegenge et al.
(a)
(b)
(c)
Fig. 7 NO induces pre-synaptic vesicle exocytosis via the cGMP
pathway. (a) Representative destaining profiles of FM1-43 after stimulation
by the NO donors SNP (1 mM) and N-Ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)
ethanamine (NOC-12) (100 lM) in
comparison with high K + . (b) The percent fluorescence intensity
remaining after 5 min stimulation with SNP was comparable with
60 mM KCl. Pre-incubation with ODQ (50 lM) for 10 min and stimulation
with SNP + ODQ for 5 min resulted in blocking of SNP-induced
FM1-43 unloading. (c) NOC-12 significantly induced unloading of
FM1-43 which was blocked by ODQ. Data represent mean ± SEM of
percent fluorescence intensity remaining after 5 min stimulation from
at least 15 synaptic puncta.
Immunocytochemical evidence for pre-synaptic maturation
of NT2 neurons was monitored by staining for the presynaptic
proteins, synapsin I, and synaptotagmin I. Synapsin
I is a member of the synapsin family specifically associated
with synaptic vesicles which has been implicated in the
synapse development and regulation of neurotransmitter
release (reviewed by de Camilli et al. 1990; Hilfiker et al.
1999; Ferreira and Rapoport 2002). Synaptotagmin I is the
best characterized isoform of the synaptotagmin family of
vesicle-associated proteins, thought to function as a calcium
sensor for fast neurotransmitter release at the synapse
(reviewed by Yoshihara and Montana 2004; Chapman 2008).
Immunofluorescence staining and quantification of synaptic
puncta showed that the level of synapsin and synaptotagmin
I increased significantly with the length of in vitro
culture. Synapsin immunoreactivity became more intense
and appeared in the neuronal process within 14–28 DIV
(Fig. 1g). Previously, for NT2 neurons generated by the
classical methods, intense immunoreactivity to synapsin was
observed upon co-culture with rat astrocytes (Hartley et al.
1999). Transcriptional up-regulation of synapsins during
retinoic acid-induced differentiation of NT2 cells has been
reported (Leypoldt et al. 2002). The immunostaining of
synaptotagmin I that appeared as punctate staining (Fig. 1h)
could indicate local accumulation of the protein at presumptive
pre-synaptic sites for participation in fast neurotransmitter
release. The expression of other pre-synaptic proteins
such as synaptobrevin, synaptophysin, and SNAP25 has also
been reported for NT2 neurons (Sheridan and Maltese 1998).
However, it remains unclear whether these pre-synaptic
proteins expressing immunopositive puncta represent functional
synaptic terminals. In this study, we showed for the
first time that human NT2 neurons display synaptic vesicle
recycling and exocytosis by two independent approaches.
Firstly, we used functional immunofluorescence methods to
label pre-synaptic terminals of NT2 neurons during synaptic
vesicle recycling. For this purpose, an antibody directed to
the luminal domain of synaptotagmin I was used in the
presence of high K + . It is well known that high K + induces
multiple rounds of exocytosis leading to labeling of the entire
pool of recycling synaptic vesicles (Klingauf et al. 1998;
Sara et al. 2002; Menegon et al. 2006). In NT2 neurons, the
labeling of synaptic vesicles by anti-luminal synaptotagmin I
depends on depolarization induced by high K + and presence
of calcium in the stimulation buffer (Fig. 2c). Our data also
show that the extent of synaptic vesicle recycling depends on
the length of in vitro culture corresponding to the expression
of the pre-synaptic proteins (Figs. 1i, j and 2b).
In the second approach, synaptic vesicle recycling and
exocytosis was analyzed by employing the fluorescent dye,
FM1-43. The protocol for labeling pre-synaptic vesicles that
display synaptic vesicle recycling by FM1-43 is well
established (Cochilla et al. 1999; Gaffield and Betz 2006).
The uptake of FM1-43Fx was significantly increased upon
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 1434–1446

Fig. 8 The cAMP/PKA pathway modulates
synaptic vesicle recycling in NT2 neurons.
The uptake of FM1-43Fx was significantly
increased in the presence of (a) forskolin
and (b) cAMP analog (8-Br-cAMP). (c) Preincubation
of NT2 neurons for 30 min with
protein kinase A antagonist (10 lM
RPcAMP or H-89) significantly reduced
forskolin induced FM1-43Fx uptake. (d)
Real time imaging of vesicle exocytosis
upon stimulation with forskolin (100 lM)
indicates fast release of FM1-43 and the
percent fluorescence intensity remaining
after 5 min was comparable with that of
high K + . Pre-incubation with H-89 (10 lM)
for 10 min and stimulation with forskolin
(100 lM) resulted in marked reduction of
the release of FM1-43. Data represent
mean ± SEM at least 80 (a–c) and 10 (d)
synaptic puncta.
stimulation with high K + in the presence of calcium. The
little punctate staining that appeared upon stimulation with
KRH (basal) during the incorporation of luminal synaptotagmin
I antibody (Fig. 2c) and FM1-43Fx (Fig. 3a) could
indicate spontaneous activity of the neurons. Intriguingly,
confocal images of NT2 neurons loaded with FM1-43Fx and
double-labeled for synaptotagmin I display clear co-localization
of the punctate staining (Fig. 4a–c). Thus, both
methods indicate that human NT2 neurons undergo synaptic
vesicle recycling in a depolarization and calcium-dependent
manner.
Unloading of FM1-43 which monitors pre-synaptic vesicle
exocytosis revealed rapid release of the dye in a both
depolarization and calcium-dependent manner (Fig. 3d–h).
This pre-synaptic vesicle exocytosis induced by high K +
presumably indicates neurotransmitter release at nerve
terminals. Recently, we have shown that NT2 neurons
express markers for glutamate, GABA, and for cholinergic
transmission (Podrygajlo et al. 2009) suggesting that these
neurotransmitters are putatively released by high K + . At the
post-synaptic site, expression of functional receptors including
GABA A-receptors (Matsuoka et al. 1997; Neelands et al.
1998, 1999;.), NMDA-type (Rootwelt et al. 1998; Paquet-
Durand and Bicker 2004; Paquet-Durand et al. 2006; Garcia
de Arriba et al. 2006), and a-amino-3-hydroxy-5-methylisoxazole-4-propionate
(AMPA)-type glutamate receptors
(Rootwelt et al. 1998) have been reported. Nevertheless,
functional synapses between NT2 neurons have been demonstrated
electrophysiologically only when cultured together
(a) (b)
(c) (d)
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 1434–1446
NO and cyclic nucleotide modulates vesicle release | 1443
with rat astrocytes (Hartley et al. 1999). Spontaneous but
uncorrelated firing patterns of NT2 neurons have been
recorded on microelectrode arrays (Görtz et al. 2004). Here,
we showed pre-synaptic vesicle release in NT2 neurons in
the absence of glia cells. Since we could demonstrate that the
neurons display increasing levels of an axonal marker (Tau),
pre-synaptic proteins (synapsin and synaptotagmin I), and
synaptic vesicle recycling with time in culture, NT2 neurons
seem to undergo pre-synaptic maturation processes. Therefore,
NT2 neurons can be utilized as a model to investigate
the cellular and molecular basis of synaptic vesicle recycling
and pre-synaptic vesicle release in human nerve cells.
NO signal transduction and pre-synaptic vesicle release
Nitric oxide synthase has been shown to be expressed during
neural development and synaptogenesis (Williams et al.
1994; Ogilvie et al. 1995; Sporns and Jenkinson 1997; Gibbs
and Truman 1998) indicating that NO is involved in synaptic
maturation processes. This study demonstrates for the first
time that NO and cyclic nucleotides regulate vesicle
recycling and pre-synaptic vesicle release in human model
neurons. Subpopulations of NT2 neurons positively stained
for the nNOS monoclonal antibody (Fig. 5b). Thus, similar
to other neurotransmitter phenotypes (Guillemain et al.
2000; Podrygajlo et al. 2009), the NT2 neurons are also
heterogeneous with respect to nNOS expression. These
neurons expressing nNOS could serve as endogenous source
of NO. The presence of functional sGC was demonstrated by
exogenous stimulation of the neurons with NO donor and

1444 | M. A. Tegenge et al.
subsequent detection of cGMP by immunofluorescence
methods (Fig. 5c–e). NO induced cGMP-IR was reduced in
the presence of a sGC inhibitor.
The NO donor, SNP significantly increased the labeling of
NT2 neurons by luminal synaptotagmin I which was
blocked by the sGC inhibitor ODQ indicating that NO
facilitates synaptic vesicle recycling via the cGMP pathway.
SNP enhanced the uptake of FM1-43Fx similar to stimulation
with high K + . The effect of exogenous NO application
was blocked by ODQ. Moreover, a membrane permeable
analog of cGMP significantly increased the uptake of FM1-
43Fx which further confirms the participation of cGMP
pathway in NO-mediated synaptic vesicle recycling. These
experimental results suggest that the NO/cGMP signaling
pathway could facilitate synaptic vesicle recycling in human
neurons. A recent study demonstrates that cGMP reduces the
cycle time for synaptic vesicles through the enhancement of
vesicular traffic rate from the recycling pool to the readily
releasable pool and accelerates fast endocytosis (Petrov
et al. 2008). Pre-synaptic exocytosis was followed in real
time during stimulation of NT2 neurons by two NO donors,
SNP and NOC-12. Both SNP and NOC-12 induced the
unloading of FM1-43 similar to high K + (Fig. 7a–c). The
unloading of FM1-43 by SNP and NOC-12 was blocked by
ODQ which indicates that NO induces vesicle release via the
cGMP pathway. Our data are in agreement with several
reports that demonstrated increased pre-synaptic transmitter
release by the NO/cGMP pathway (Arancio et al. 1995,
2001; Lu et al. 1999; Wildemann and Bicker 1999; Li et al.
2004; Nickels et al. 2007). To analyze whether the NO/
cGMP induced enhancement of vesicle exocytosis is caused
by excitatory effects at the level of the membrane or by
directly facilitating the calcium-dependent release machinery,
electrophysiological techniques will be required. Moreover,
in hippocampal neurons and synaptosomes, NO has
been shown to induce neurotransmitter release independent
of intracellular calcium levels (Meffert et al. 1994; Sporns
and Jenkinson 1997).
One potential intracellular downstream effector protein for
the cGMP signaling pathway is PKG. Enhanced transmitter
release via phosphorylation of pre-synaptic voltage-gated K +
channels by PKG has recently been implicated (Yang et al.
2007). Additionally, formation of new cluster of pre- and
post-synaptic proteins which involves phosporylation of PKG
substrate proteins, vasodilator-stimulated phosphoprotein,
and RhoA have been reported (Wang et al. 2005). A direct
action of NO without the involvement of cGMP and PKG via
S-nitrosylation on the exo–endocytotic machineries has been
also implicated (Meffert et al. 1996; Ahern et al. 2002). Even
though our data from both immunofluorescence and FM
imaging strongly support the participation of the cGMP
pathway, we could not completely exclude the direct action of
NO since the sGC inhibitor, ODQ did only partially block the
unloading of FM1-43 induced by NOC-12 (Fig. 7c).
Pre-synaptic activation of calcium-sensitive adenylyl
cyclase that leads to a rise in the level of cAMP and
consequent activation of PKA has been implicated to
enhance the probability of neurotransmitter release (Siegelbaum
et al. 1982; Chavez-Noriega and Stevens 1994;
Trudeau et al. 1996; Evans and Morgan 2003). The proteins
involved in pre-synaptic vesicle exocytosis have been
suggested as major substrates for PKA-dependent phosphorylation
(reviewed by Leenders and Sheng 2005). Phosphorylation
of synapsin by the cAMP/PKA signaling which leads
to release of vesicles from the reserve pool has been
suggested to enhance pre-synaptic vesicle releases (Fiumara
et al. 2004, 2007; Bonanomi et al. 2005; Menegon et al.
2006).
Here, we showed that stimulation of adenylyl cyclase by
forskolin induces vesicle exocytosis and facilitates vesicle
recycling while inhibition of PKA by Rp-cAMP or H-89
blocks the effect of forskolin indicating that the cAMP/PKA
pathway modulates vesicle release and synaptic vesicle
recycling in NT2 neurons. Our result is in line with the
increased neurotransmitter release reported for hippocampal
neurons upon activation of cAMP/PKA signaling (Trudeau
et al. 1996, 1998). However, we do not know at present
which target proteins are modified by NO and cyclic
nucleotide signaling in NT2 neurons. In our readily accessible
human model neurons, we can now investigate the
candidate proteins modulated via NO and cyclic nucleotide
signal transduction.
In summary, we have presented a model of human neurons
that express increasing levels of pre-synaptic proteins and
display synaptic vesicle recycling. Our data also revealed for
the first time that the NO and cyclic nucleotide signal
transduction modulates synaptic vesicle recycling and exocytosis
in human model neurons.
Acknowledgements
We thank Dr J. de Vente for his kind gift of the cGMP antiserum, Dr
S. Knipp for the discussion during the preparation of the manuscript
and S. Tan for technical support. This research was in part supported
by BMBF grant 0313732 to G. Bicker and by a DFG grant (FG
1103, BI 262/16-1). M. A. Tegenge received a Georg-Christoph-
Lichtenberg scholarship from the Ministry for Science and Culture
of Lower Saxony.
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Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 1434–1446

Nitric oxide signaling as regulator of human neuronal progenitor cell migration
Million Adane Tegenge 1,2 , Thomas Dino Rockel 3 , Ellen Fritsche* 3,4 , Gerd Bicker* 1,2
1. Division of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover,
Bischofsholer Damm 15, D-30173 Hannover, Germany
2. Center for Systems Neuroscience (ZSN), Hannover, Germany
3. Group of Toxicology, Institut für Umweltmedizinische Forschung gGmbH at the Heinrich Heine-
University, Düsseldorf, Germany
4. Clinic of Dermatology, RWTH Aachen, Germany
*Corresponding authors: Gerd Bicker, Division of Cell Biology, Institute of Physiology, University
of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany, Tel.:
+49 5 11 856 7765, Fax: +49 5 11 856 7687
e-mail: gerd.bicker@tiho-hannover.de
Ellen Fritsche, Group of Toxicology, Institut für Umweltmedizinische Forschung gGmbH at the
Heinrich Heine-University, Auf’m Hennekamp 50, 40225, Düsseldorf, Germany, Tel. +49
2113389217, Fax: +49 211 3190910
(In preparation)
e-mail: ellen.fritsche@uni-duesseldorf.de

Abstract
Neuronal migration is a cellular event that is critical for the development of functional neuronal
network. A number of genes responsible for human congential disorders featuring neuronal
migration defects have been identified. In this study we examine the role of nitric oxide (NO)
signaling in the migration of fetal human neural progenitor cells (hNPCs) which was cultured as
neurospheres. Cells migrate out of the human neurospheres and differentiate into both neuronal and
glial cells. The migrating cells express functional guanylyl cyclase (sGC) that produces cGMP upon
activation with NO, an effect reduced in the presence of sGC inhibitor. Application of enzyme
inhibitors of sGC and protein kinase G (PKG) blocked the migration of cells out of human
neurospheres. Inhibition of sGC can be rescued by a membrane permeable analogue of cGMP. In
gain of function experiments both NO donor and cGMP analogue facilitate cell migration
suggesting that NO-cGMP signaling positively regulates hNPCs migration. These results provide
first experimental evidence for a role of NO-cGMP signal transduction as a regulator of cell
migration during early development of the human brain.
Key words: brain development, human neurospheres, NO, cGMP
2

Introduction
Neuronal precursor cell migration during early phase of development and in some area of the adult
brain is important for the formation of functional networks in the brain. The gaseous messenger
nitric oxide (NO), which is produced by nitric oxide synthase enzymes (NOS), appears to play
critical role in proliferation, migration and synaptogenesis during nerve cell development
(Enikolopov et al., 1999; Cárdenas et al., 2005; Bicker, 2005). A major receptor for NO is the
cGMP synthesising enzyme soluble guanylyl cyclase (sGC) (Garthwaite, 2008). NO signal
transduction has been shown to regulate the migration of cerebellar neurons, insect enteric neurons,
and early developing Xenopus neuronal cells (Tanaka et al., 1994; Haase and Bicker, 2003;
Peunova et al., 2007; Knipp and Bicker, 2009). Neuroanatomical studies using markers against
NOS and sGC indentify the migrating neuroblasts of the rostral migratory stream as potential
targets for NO signaling in the adult brain (Moreno-López et al., 2000; Gutierrez-Mecinas et al.,
2007). In a model of ischemic stroke, NO donor has been shown to facilitate neuroblasts migration
in subventricular zone and dentate gyrus (Zhang et al., 2001; Cui et al., 2009).
The transient expression of NOS during development has provided additional evidence for an
involvement of NO signaling in neuronal migration. In the developing rat brain, nNOS has been
demonstrated to be expressed transiently, reaching the highest level between embryonic day 16 and
postnatal day 0. These periods corresponds to the migration of neuronal precursors from the
ventricular zone to the external layers of the cortex (Bredt and Snyder, 1994; Nott et al., 2008). In
the fetal human spinal cord, some neurons express NOS as they migrate to their final destination
(Foster and Phelps, 2000). Recently, we have used cultured human NT2 cells to demonstrate that
NO and cGMP signal transduction regulate the migration of neuronal precursors (Tegenge and
Bicker, 2009). However, there is no functional evidence indicating the participation of NO signaling
during early development of human nervous system.
The expansion of multi-potent human neural progenitor cells in vitro may offer a model system to
study the molecular and cellular basis of human brain development. In this study we used human
neural progenitor cells (hNPCs) maintained as three-dimensional free-floating neurospheres, to
investigate the role of NO/cGMP signal transduction in neuronal migration. We used
immunocytochemistry to show that hNPCs express neuronal NOS (nNOS) and functional sGC. By
employing specific enzyme activators and inhibitors we provide the first experimental evidence that
NO-cGMP signal transduction regulates cell migration in developing human brain cells.
Material and Methods
NOC-18 [2, 2-(Hydroxynitrosohydrazino) bis-ethanamine] was purchased from Calbiochem
(Darmstadt, Germany), 8-Br-cGMP (8-Bromoguanosine 3’, 5’-cyclophosphate) and Rp-8-Br-cGMP
3

(8-Bromoguanosine-3’, 5’-cyclophosphothioate, Rp-isomer) were purchased from Alexis
Biochemicals (Lörrach, Germany). All other substances were obtained from Sigma (Taufkirchen,
Germany) unless otherwise noted.
Cell culture
The hNPCs used in this study were purchased from Lonza Verviers SPRL (Verviers, Belgium),
which was obtained from three individuals. The hNPCs was cultured as previously described
(Fritsche et al., 2005). Briefly, cells were cultured in proliferation medium (Dulbecco´s modified
Eagle medium and Hams F12 (3:1) supplemented with B27 (Invitrogen GmBH, Karlsruhe,
Germany), 20 ng/ml epidermal growth factor (EGF) (Biosource, Karlsruhe, Germany), 20 ng/ml
recombinant human fibroblast growth factor (rhFGF) (R&D Systems, Wiesbaden-Nordenstadt,
Germany), 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified 92.5% air/7.5% CO2
incubator at 37°C in suspension culture. Medium was changed every 2–3 days and spheres were
chopped with a McIlwaine tissue chopper. Differentiation was induced by growth factor withdrawal
in the presence of DFN medium (Dulbecco modified Eagle medium and Hams F12 (3:1)
supplemented with N2 (Invitrogen)) upon culturing the neurospheres on chamber slides (Becton
Dickinson, Bedford, MA) coated with poly-D-lysine and laminin.
Migration assay
hNPCs were seeded on poly-D-lysine and laminin coated chamber slides at density of five
neurospheres. The culture was treated with chemicals diluted in the differentiation medium for at
least 72 h with a medium change after 48 h. The NO donor NOC-18, which decays with long half
life (about 57 h at 22 o C) was prepared as 100 mM stock solution in 10 mM NaOH solution. ODQ
(1H-[1, 2, 4]-oxadiazolo[4,3-a]quinoxalin-1-one) was prepared as 20 mM stock solution in DMSO
(dimethylsulphoxide). 8-Br-cGMP and Rp-8-Br-cGMP were directly dissolved in the medium. To
determine the migration of cells out of neurospheres, images were acquired after 24 h of chemical
application and migration distance was measured at four distinct locations from the rim of the
spheres to furthest migrated cells (Moors et al., 2007; Tegenge and Bicker, 2009). The data were
presented as mean ± s.e.m of three independent experiments.
LDH cytotoxicity assay
The lactate dehydrogenase (LDH) assay (CytoTox-One, Promega, Mannheim, Germany) was
employed to assess cell death by measuring the amount of LDH which is released into the medium
from dead cells. The assay was performed according to the manufacturer’s instructions. Briefly,
following exposure to the chemicals for 48 h, supernatants of the culture medium (100 µl) was
4

mixed with 50 µl of Cyto-Tox-One reagent and kept for 4 h in 96- well plate. Fluorescence intensity
was detected at excitation/emission wavelength of 540/590 nm. Results were presented as mean ±
s.e.m of percent maximum LDH released from four measurements.
Immunocytochemistry
Immunocytochemical detection of βIII-tubulin (Sigma 1:10,000), nestin (Calbiochem 1:400) and
glial fibrillary acidic protein (GFAP, Sigma 1:500) was performed as previously described
(Tegenge and Bicker, 2009). For the detection of cGMP immunoreactive (cGMP-IR) cells, hNPCs
were preincubated for 20 min at 37 o C with 1 mM sodium nitroprusside (SNP) as a NO donor, 20
µM YC-1 (3-(50-Hydroxymethyl-20-furyl)-1-benzyl indazole) as an enhancer of NO-induced
activity of sGC, 50 µM of ODQ as sGC inhibitor and 1 mM IBMX (3-isobutyl-1-methylxanthine)
as phosphodiesterase inhibitor. Cultures were washed once with PBS and fixed with 4% PFA. The
polyclonal sheep cGMP antiserum (1:10,000; a kind gift from Dr. J. de Vente, Maastricht
University, Netherlands) was used as primary antibody to detect the level of cGMP.
Microscopy and Data analysis
Preparations were examined with a fluorescent microscope Zeiss Axiovert 25 equipped with an
Axiocam 3900 digital camera or Zeiss Axio Observer D1 (Zeiss, Göttengen, Germany). The
percentage of nestin, βIII-tubulin, GFAP and cGMP-IR cell bodies was determined dividing the
number of positively stained cells by the total number of cells in the migration area. The data were
presented as mean ± s.e.m of at least two independent experiments. Statistical comparisons of
control versus treatment were performed with the unpaired two-tailed Student’s t-test. Levels of
significance are indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.
Results
Nitric oxide-sensitive sGC is expressed in early developing human brain cells
On western blots a monoclonal antibody against the nNOS labelled a major protein band around
155 KDa (Fig. 1), indicating that nitric oxide could be endogenously produced early during the
development of human brain. To reveal functional sGC that synthesizes cGMP upon activation by
NO, we used an antibody against cGMP (de Vente et al., 1987). In the absence of exogenous NO,
only few cGMP-IR cells were detected (Fig. 2 A & D). The number of cGMP-IR cells increased
significantly up to 38.9 ± 2% upon stimulation with the NO donor SNP (Fig. 2 B & D). The NO
induced cGMP-IR cells were significantly reduced when SNP stimulation was accompanied by the
sGC inhibitor ODQ (Fig. 2 C & D).
5

Fig. 1 The antibody against nNOS recognizes a protein band of apparent molecular weight at 155
kDa from the proliferating hNPCs on Western blot. NT2 spherical aggregates treated with retinoic
acid for 7 days was used as a positive control. The lower band represents acetylated-α-tubulin.
Fig. 2 Functional NO/cGMP signaling is present in early developing human nerve cells. The
presence of NO-sensitive functional sGC was demonstrated by immunocytochemical detection of
cGMP. hNPCs were exposed for 20 min to (A) 1 mM IBMX + 20 µM YC-1 (control), (B) SNP (1
mM) or (C) SNP (1mM) + ODQ (50 µM) together with IBMX + YC-1. Cultures were fixed with
4% PFA and stained against cGMP. (D) The cGMP-IR cells were counted after stimulation with
NO donor (SNP) and compared with the control. (E) Schematic drawing of NO/cGMP signal
6

transduction together with an array of activators and inhibitors of the pathway. Scale bars: 50 µm.
Data are mean ± s.e.m of 10 neurospheres from at least two independent experiments.
cGMP-IR nestin and GFAP positive cells direct the migration of βIII-tubulin-IR cells
The progress of neuronal differentiation within hNPCs was monitored by immunocytochemical
staining against cytoskeletal markers of progenitor cells (nestin), early neurons (βIII-tubulin) and
glial cells (GFAP). After 72 h of migration, the majority of the cells that migrate out of the
neurospheres stained for nestin with a few interspersed neurons (Fig. 3A). A major subset of the
cells was positive for the glial marker GFAP (Fig. 3A). Co-staining of cGMP with cytoskeletal
markers indicated that about 53.5 ± 2 % was GFAP-IR while the nestin and βIII-tubulin-IR cells
constitute about 21 ± 3 and 9.6 ± 1 %, respectively (Fig. 3 B-D). Cells at the forefront of migration
were mainly composed of the nestin and GFAP positive which are double-labeled for cGMP (Fig. 3
E- G).
cGMP/PKG signaling positively regulates the migration of hNPCs
After showing the presence of a functional NO signal transduction pathway in the hNPCs we tested
whether cGMP/PKG signaling modulates the migration of cells out of the human neurospheres. The
concentration of both inhibitors and activators of the pathway (Fig. 2 E) were selected based on
previous experiments using migrating NT2 cells (Tegenge and Bicker, 2009). The sGC inhibitor
(ODQ) significantly reduced the migration of cells out of neurospheres (Fig. 4 A, B). In the
presence of PKG-I inhibitor (Rp-8-Br-cGMP), the migration of cell was reduced in a dose
dependent manner (Fig. 4 C). Application of a cell membrane permeable analogue of cGMP, 8-BrcGMP
partially rescued the blocking of cell migration by ODQ (Fig. 4 A, D). LDH cytotoxicity
assay was conducted after the application of enzyme inhibitors of sGC and PKG. Both ODQ and
Rp-8-Br-cGMP did not alter the viability of cells at the concentration used to inhibit cell migration
(Fig. 4 E, F).
7

Fig. 3 Immunocytochemical characterization of hNPCs for cytoskeletal markers and NO signal
transduction. (A) After 72 h of migration on laminin coated chamber slides cells that migrate out of
human neurospheres express mainly nestin and GFAP with few interspersed βIII-tubulin-IR cells.
After exposure of hNPCs to SNP + IBMX + YC-1 for 20 min culture was co-stained for cGMP and
(B) nestin, (C) βIII-tubulin or (D) GFAP. (E-G) cGMP-IR cells at the forefront of migration were
co-stained for nestin or GFAP but not for βIII-tubulin. Arrow heads (B-G) show representative colocalization
of cGMP with respective cytoskeletal markers. Dense cellular aggregate staining (A-D)
delineates the edge of the neurospheres. Scale bar: 50 µm.
NO and cGMP facilitates the migration of hNPCs
Exposure of hNPCs to the NO donor NOC-18 (1-10 µM) for 24 h significantly facilitated the
migration of cells out of the spheres (Fig. 5 A, B). However, 100 µM of NOC-18 significantly
inhibited cell migration (Fig. 5 A, B). Parallel to cell migration experiments, we conducted cell
viability assay upon NOC-18 application, which showed no sign of cytotoxicity (Fig. 5 C). A
significant reduction of NO-induced facilitation of cell migration was observed when NOC-18 was
used in combination with ODQ or Rp-8-Br-cGMP (Fig. 5 D). Application cGMP analogue, 8-BrcGMP
significantly facilitated the migration of cells (Fig. 5 E).
Finally, we counted the number of βIII-tubulin-IR and GFAP-IR cells to examine the effects of
bioactive chemicals that activate and inhibit the sGC/PKG pathway. At the concentration used to
8

modulate cell migration, none of the chemicals significantly altered the proportion of neuronal and
glial marker (Fig. 6).
Fig. 4 The sGC/PKG enzyme inhibitors block the migration of hNPCs. (A) Representative
photomicrograph of hNPCs after 24 h of migration under control condition (0.25% DMSO) and in
the presence of ODQ or 8-Br-cGMP + ODQ. (B-D) Migration distance from the rim of the sphere
to the furthest migrated cells was quantified at four distinct locations per sphere. Application of (B)
ODQ and (C) Rp-8-Br-cGMP significantly blocked the migration hNPCs. (D) The blocked of cell
migration by ODQ (50 µM) was rescued by 8-Br-cGMP (1 mM). Both (E) ODQ and (F) Rp-8-BrcGMP
did not cause cytotoxicity. Data represent mean ± s.e.m of at least 10 neurospheres (B-D)
and four measurements (E & F). Scale bar: 500 µm
9

Fig. 5 NO donor and cell membrane permeable analogue of cGMP facilitates cell migration. (A)
Representative photomicrographs of hNPCs after 24 h of migration under control condition and in
the presence of 10 µM NOC-18 and 100 µM NOC- 18. (B) The migration of cells increased
significantly in the presence of 1-10 µM of NOC-18. (C) The viability of cells was not altered by
NOC-18. (D) Co-application of NOC-18 (10 µM) together with down-stream enzyme inhibitor of
sGC (ODQ, 50 µM) or PKG (Rp-8-Br-cGMP,1 µM) reduced NO-induced migration of cells. (E) 8-
10

Br-cGMP facilitated cell migration. Data represent mean ± s.e.m of 15 neurospheres (B, D, & E)
and four measurements (C). Scale bar: 500 µm
Fig. 6 The proportion of neuronal and glial cells was not changed in the presence of chemicals that
modulate cell migration. Application ODQ (50 µM), Rp-8-Br-cGMP (1 µM), NOC-18 (10 µM) and
8-Br-cGMP (1 mM) for 72 h in hNPCs culture did not significantly alter the percentage �III-tubulin
and GFAP-IR cells. Data are mean ± s.e.m. of 10 neurospheres from at least two independent
experiments.
Discussion
hNPCs as a model for early developing human brain
When the proliferating hNPCs were plated on laminin coated substrates, cells migrate out of the
neurospheres. The migration distance from the rim of the spheres to the furthest migrated cells
increases with time in vitro and accompanied by differentiation into neuronal and glial cells
(Fritsche et al., 2005; Moors et al., 2007; 2009). Here, we performed immunocytochemical staining
against cytoskeletal markers of progenitor cells, early neurons and glial cells. The nestin and GFAP
positive cells constitute the major proportion of cells that migrate out of the neurosphere within 72 h
(Fig. 2 A). Nevertheless, upon prolonged time of culturing (7-14 days) most of the nestin positive
progenitor cells have been demonstrated to differentiate into βIII-tubulin-IR cells (Brannen and
Sugaya, 2000; Moors et al., 2009). Since hNPCs generate both neuronal and glial cells with the later
in a higher proportion and at the forefront of migration (Fig. 3 G), the neurons appear to migrate
along a glial scaffold. Indeed, real-time microscopic observation for 24 h revealed that cells migrate
out of human neurosphere mainly by radial migration (Moors et al., 2009). Thus, the use of hNPCs
to study cell migration may mimic aspects of radial neuroblast migration which accounts for 80-90
% of cell migration during early development of brain (Ayala et al., 2007).
11

NO/cGMP/PKG signaling mediate the migration of hNPCs
Several studies implicate the involvement of NO-cGMP signaling in neuronal migration during
nervous system development (Tanaka et al., 1994; Haase and Bicker, 2003; Peunova et al., 2007;
Knipp and Bicker, 2009) and after CNS injury in adults (Zhang et al., 2001; Cui et al., 2009;
Gutierrez-Mecinas et al., 2007). In this study we provide the first experimental evidence that NOcGMP
signaling positively regulate the migration of fetal human neuronal progenitor cells. Firstly,
we showed expression of NO-sensitive sGC in cells that migrate out of human neurospheres by
immunocytochemical detection of cGMP. Upon stimulation with NO donor the number of cGMP-
IR cells increased significantly, which was reduced in the presence of sGC inhibitor. Thus, our data
clearly demonstrate for the first time to our knowledge that NO-sensitive sGC is present early
during human brain development. Co-staining of cGMP with cytoskeletal markers indicated that the
major proportion of the cGMP producing cells were GFAP-IR with a lower percentage of nestin
and βIII-tubulin-IR cells. A close examination of the forefront of migration revealed mainly nestin
and GFAP positive cells which are double-labeled for cGMP (Fig. 3 E-G). These migratory nestin
and GFAP positive cells are trailed by βIII-tubulin-IR neuronal cells (Fig. 3 C & F).
Application of enzyme inhibitors of the sGC/PKG pathway in hNPCs culture significantly reduced
the migration of cells out of the neurospheres (Fig. 4 A-C). This loss of function effect and the
presence of cGMP-IR cells at the forefront of migration (Fig. 3 E-G) infer that a certain level of
cGMP is required to facilitate the migration of cells out of the neurospheres. Morphological
observation of neurospheres and LDH assay revealed no sign of cytotoxicity caused by sGC/PKG
inhibitors. Moreover, a cell membrane permeable analogue of cGMP, 8-Br-cGMP partially rescued
the blocking of cell migration by ODQ (Fig. 4 A, D). Thus, it is unlikely that unspecific side effects
of the chemical blocker contribute to inhibition of cell migration.
In a gain of function experiments, exposure of hNPCs to the NO donor NOC-18 (1-10 µM) and
cGMP analogue (8-Br-cGMP) significantly facilitated the migration of cells out of the neurospheres
(Fig. 5 A, B, E). This gain of function experiment is in line with our recent finding on the retinoic
acid induced differentiating human neuronal precursor (NT2) cells (Tegenge and Bicker, 2009). In
NT2 cells, NOC-18 facilitated cell motility in a concentration range of 1-100 µM, above which we
observed deleterious effect on cell viability and proliferation (Tegenge and Bicker, 2009). Here, a
concentration of 100 µM of NOC-18 significantly inhibited cell migration (Fig. 5 A, B) without
altering cell viability (Fig. 5 C). The inverted U-shaped dose-response curve (Fig. 4B) suggests that
NO is playing a dual role, resulting in a facilitation of cell migration at low concentration while
slowing down at higher concentrations. Similar concentration dependent effects on growth cone
12

motility have been observed on developing snail neurites (Trimm and Rehder, 2004) and migrating
neutrophils (Elferink and VanUffelen, 1996).
To directly link the effect of NO with its potential downstream target enzymes, NO donor was co-
applied with sGC or PKG inhibitors. A significant reduction of NO-induced facilitation of cell
migration was observed when NOC-18 was used in combination with ODQ or Rp-8-Br-cGMP (Fig.
5 D). This provides further evidence for the involvement of cGMP and PKG in NO mediated
neuronal progenitor cell migration. The detailed mechanism by which cGMP activated PKG
facilitates cell motility is presently unknown. In both neuronal and non-neuronal cells
reorganization of actin cytoskeleton is caused downstream of NO/cGMP pathway (Brown et al.,
1999; Haase and Bicker 2003, Borán and García, 2007), which possibly involve inhibition of RhoA
GTPase (Sawada et al. 2001; Gudi et al. 2002; Borán and García 2007). The PKG substrate
Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family proteins that regulate actin
polymerization have been also suggested to mediate the action of cGMP during cell motility
(Sporbert et al., 1999; Chen et al., 2008; Lindsay et al., 2007).
A concentration gradient of endogenously produced NO has been proposed to regulate cell-cycle
progression in the developing chicken neural tube (Traister et al., 2002). Moreover, during early
development of Xenopus NO has been shown to differentially coordinate cell proliferation and
motility (Peunova et al., 2007). Likewise, in the intact fetal human brain gradients of the diffusible
messenger NO may be essential to coordinate the distinct processes of cell proliferation,
differentiation and motility.
Acknowledgments
We thank Dr J. de Vente for his kind gift of the cGMP antiserum, Dr. M. Stern for the discussion
during the preparation of the manuscript and Nadine Seiferth for technical support. M.A. Tegenge
received a Georg-Christoph-Lichtenberg scholarship from the Ministry for Science and Culture of
Lower Saxony. This work was supported by the BMBF grants (013925C) to E. Fritsche, (013925D)
to G. Bicker and DFG grant (FG 1103, BI 262/16-1).
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15

Declaration
I herewith declare that I autonomously carried out the PhD-thesis entitled
“Nitric Oxide Mediated Signal Transduction in Networks of Human Model Neurons”.
I did not receive any assistance in return for payment by consulting agencies or any other
person. No one received any kind of payment for direct or indirect assistance in correlation to
the content of the submitted thesis.
I conducted the project at the following institutions:
Division of Cell Biology, Institute of Physiology, University of Veterinary Medicine
Hannover, Germany.
The thesis has not been submitted elsewhere for an exam, as thesis or for evaluation in a
similar context.
I hereby affirm the above statements to be complete and true to the best of my knowledge.
_______________________________
Million Adane Tegenge