Characterization of the Role of Rac3 in Neuronal Function

Gualdoni, Sara (2008). Characterization of the Role of Rac3 in Neuronal Function. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f273

Abstract

A major goal in neurobiology is to study the mechanisms by which neurites navigate towards their targets to establish functional synapses. While a large amount of information is available on the extra cellular signals driving neuritogenesis and synaptogenesis, major gaps exist in the comprehension of the intracellular events.

Rho family GTPases regulate the dynamic of the actin cytoskeleton and plays a crucial role in regulating neuronal development. In particular, Rac has been implicated in axon elongation and guidance, in dendritic growth and in the regulation of dendritic spines formation and plasticity. There are three Rac genes in mammals: Rac1, Rac2 and Rac3, sharing about 90% protein identity, all participating in several functions, including actin dynamics and adhesion (Hall, 1998). Rac1 is ubiquitous, while Rac2 is specific to the hematopoietic lineage and Rac3 is developmentally regulated in the vertebrate brain (Corbetta et al., 2005), with the highest expression during neurite branching and synaptogenesis (Bolis et al., 2003). The co-expression of Rac1 and Rac3 in the vertebrate nervous system suggests that they are needed to solve individual developmental and maintenance issues in the nervous system.

The aim of my work is to analyse the function of Rac1 and Rac3 during nervous system development. I took advantage of both in vitro and in vivo approaches to answer this question. Comparison between wild-type and Rac3-/- hippocampal neurons showed no differences in neuritogenesis and synaptogenesis processes. On the other hand, Rac1 depletion by RNAi in both wild-type and Rac3-/- hippocampal neurons during early stage of neuronal development showed specific alteration of the dendritic development without affecting axonal outgrowth. Reduced Rac1 levels impaired growth cone dynamics and F-actin levels in both neurites and growth cones. These results show that endogenous Rac1 rather than Rac3 activity is essential for early dendritic development, but not required for the initial establishment of neuronal polarity and axonal development. Depletion of either Rac1 or Rac3 during later stages of in vitro neuronal development did not severe dendritic spines morphogenesis, while double depletion strongly impaired the formation and maturation of dendritic spines. All together these in vitro results reinforce the hypothesis that Rac1 and Rac3 are both important for neuronal development underlying a specific role for Rac3 in later events of neuronal development.

We next evaluated the effects of the depletion of Rac1 and Rac3 in vivo. I generated mice with conditional deletion of Rac1 in neurons (Rac1N), by crossing Rac1Flox/Flox mice (Walmsley et al., 2003) with transgenic Synapsin-Cre mice (Zhu et al., 2001) and double RaclN/Rac3-/- mice.

As Rac3-/-, also Rac1N developed normally and was fertile. Both Rac3-/- and Rac1N did not reveal major abnormalities in the brain cytoarchitecture, cell layering or alteration in the general organization of dendrites and synapses. Double RaclN/Rac3-/- mice died around 13 days after birth (P13) and showed strong neurological defects. Morphological analysis revealed evident alterations in the hippocampus. The abnormalities in the organization of the hippocampus may be correlated with the frequent spontaneous epileptic seizures developing after P7 in these mice. We found that double depletion of Rac1 and Rac3 induced an increase of WAVE1 (Ser310) and PAK1 (Thr423) phosphorylation, and reduced the total levels of RhoA. All together the data obtained in this thesis show for the first time that both Rac1 and Rac3 are important for neuronal development, while mutation of each gene leads to viable animals with no evident anatomical defects due to partial compensatory effects.