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

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
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