Neuritogenesis, the first step of neuronal differentiation, is characterized by breakage of the initial, approximately spherical symmetry as nascent neurites emerge from the soma (Da Silva and Dotti, 2002
). These processes extend steadily until one (the future axon) starts growing more rapidly, inducing morphological polarization. As neurites extend further and acquire their final axonal or dendritic identities, neurons establish synaptic contacts and reach full maturation.
Different in vivo and in vitro studies over the last 30 yr have hinted at the importance of neuritogenesis. As neurons start migrating in the developing brain, they form new leading edges that develop into short extensions and operate as guides for migration (Hatten, 1999
). Such extensions sprout as the early neurons contact particular extracellular environments (Baum and Garriga, 1997
), and failure to do so precludes proper migration (Gleeson and Walsh, 2000
). Thus, neuritogenesis is fundamental for initiating migration and patterning and, ultimately, for the inception of neuronal differentiation.
The intracellular events triggering neurite sprouting are not established. However, regulation of actin dynamics via particular signaling pathways is known to be a crucial mechanism directing the dramatic morphological changes observed in subsequent differentiation stages (Luo, 2002
). One such group of actin-regulating pathways is directed by Rho small GTPases, such as RhoA (Luo, 2000
). Overexpression of constitutively active RhoA has been shown to induce neurite retraction and arrest growth in neuronal cell lines (Jalink et al., 1993
; Kozma et al., 1997
) and in primary neuronal populations (Bito et al., 2000
). Conversely, direct inactivation of RhoA by ADP-ribosylation (Sekine et al., 1989
) using the C3-exoenzyme (a specific RhoA inhibitor) enhances neurite extension and growth cone movement (Jalink et al., 1994
; Hirose et al., 1998
). Likewise, inactivation of the RhoA kinase ROCK (a well-characterized downstream effector of RhoA) produces a similar effect in cerebellar granule cells (Bito et al., 2000
). These findings suggest that the capacity of RhoA-directed pathways to control actin stability is fundamental to events such as neurite elongation, guidance, and branching.
Despite the fact that RhoA can convey information to the actin cytoskeleton during these developmental steps via cofilin (Bamburg, 1999
), other actin-binding proteins, such as the profilins, may be involved as well. Profilins have been implicated in the maintenance of cytoskeletal integrity in a variety of organisms such as Dictyostelium discoideum
(Haugwitz et al., 1994
), yeast (Haarer et al., 1990
), and Drosophila melanogaster
(Verheyen and Cooley, 1994
). In the fly, the sole form of profilin (chickadee
) has been shown to play a role in motor neuron axon extension as chickadee
mutations arrest growth (Wills et al., 1999
). In mammals, there are different profilin isoforms that, while sharing similar biochemical properties, have diverse tissue distributions. Profilin I (PI) is ubiquitous, whereas both profilin II isoforms (PIIa and PIIb) are largely restricted to the brain (Witke et al., 2001
). A third profilin has been described recently and its expression detected exclusively in kidney and testis (Hu et al., 2001
). Apart from the fact that PIIa is a brain-specific profilin isoform, the only actin-binding protein specifically interacting with the RhoA downstream kinase ROCK in brain extracts is PIIa (Witke et al., 1998
). This led us to hypothesize that PIIa may be a key player in neuronal-specific events directed by the RhoA–ROCK pathway.
In this work, we show that RhoA and its specific downstream effector ROCK are essential during neuritogenesis in mammalian hippocampal neurons by modulating actin stability. Consistent with the aforementioned hypothesis, such modulation is dependent on the downstream effector profilin IIa, a protein of previously undetermined function. Furthermore, we show that this early neuronal program is regulated by different physiological stimuli. These findings reveal a central regulatory mechanism directing the, as yet, uncharacterized process of initial neuronal symmetry breakage.