Different Roles for RhoA During Neurite Initiation, Elongation, and Regeneration in PC12 Cells

Abstract

Abstract:The goal of the present study was to characterize the effects of RhoA at different stages of nerve growth factor (NGF)-induced neuronal differentiation in the PC12 model. This comparative analysis was prompted by previous studies that reported apparently opposite effects for Rho in different models of neuronal differentiation and regeneration. PC12 cells were transfected with activated V14RhoA or dominant negative N19RhoA under the control of either a constitutive or a steroid-regulated promoter. Upon exposure to NGF, V14RhoA cells continued to proliferate and did not extend neurites; however, they remained responsive to NGF, as indicated by the activation of extracellular signalregulated kinases. This inability to differentiate was reversed by C3 toxin and activation of cyclic AMP signaling, which inactivate RhoA. N19RhoA expression led to an increase in neurite initiation and branching. In contrast, when the RhoA mutants were expressed after NGF priming, only the rate of neurite extension was altered; V14RhoA clones had neurites approximately twice as long, whereas neurites of N19RhoA cells were ϳ50% shorter than those of appropriate controls. The effects of Rho in neurite regeneration mimicked those observed during the initial stages of morphogenesis; activation inhibited, whereas inactivation promoted, neurite outgrowth. Our results indicate that RhoA function changes at different stages of NGF-induced neuronal differentiation and neurite regeneration.

The gene for a new brain specific RhoA exchange factor maps to the highly unstable chromosomal region 1p36.2–1p36.3.

“…Since GEF720 expression is brain speci®c, we analysed its activity in PC12 neuron-like cells. In this system, it has been reported previously that active RhoA inhibits neurite outgrowth triggered by NGFtreatment (Sebok et al, 1999). In the presence of serum, PC12 cells expressing GEF720 maintained the same morphology as untransfected cells (Figure 6b, panels a and b).…”

“…Our understanding of these RhoA dependent signaling cascades relies on the identi®cation of the speci®c upstream activators of the exchange factors. In the case of neuronal di erentiation, RhoA appears to be involved in di erent steps of NGF-induced neuronal di erentiation (Sebok et al, 1999) suggesting that di erent RhoA GEFs may be sequentially active or that the same GEF may be activated through di erent cascades. Identifying the proteins that interact with GEF720, a GEF speci®cally expressed in adult and fetal brain, should provide new insights in understanding the role of RhoA in controlling neural di erentiation pathways.…”

LIM Kinase 1 Activates cAMP-responsive Element-binding Protein during the Neuronal Differentiation of Immortalized Hippocampal Progenitor Cells

“…In addition, the Rho-associated kinase-dependent pathway is reported to suppress nerve growth factor-induced Rac activation and the neurite outgrowth of PC12 cells (55,56). Moreover results obtained from PC12 cells are not generally applicable in CNS hippocampal neuronal cells, strongly suggesting that mitogenic and differentiating signals are transmitted through distinct pathways within peripheral nervous system-derived PC12 and hippocampal cells, respectively.…”

Neuronal Differentiation

“…Profilin I R136D mutant cells do not respond to inhibition of ROCK activity Changes in actin dynamics during neurite outgrowth are governed by multiple signal transduction pathways, many of which converge on the Rho GTPases (Sebok et al, 1999;Nikolic, 2002). Constitutive active RhoA causes neurite retraction, while inhibition of RhoA enhances neurite extension (Jalink et al, 1994;Kozma et al, 1997).…”

“…7). In the early phase of NGF stimulation, PLC␥1 is activated, causing a local drop in PtdIns(4,5)P 2 levels, and the Rho-ROCK pathway is inhibited (Kim et al, 1991;Sebok et al, 1999). Profilin I and IIa, amongst many other proteins, are free to interact with actin (and their proline-rich ligands), thereby stimulating neurite formation.…”