Generation and characterization of Rac3 knockout mice.

Rac proteins are members of the Rho family of GTPases involved in the regulation of actin dynamics. The three highly homologous Rac proteins in mammals are the ubiquitous Rac1, the hematopoiesis-specific Rac2, and the least-characterized Rac3. We show here that Rac3 mRNA is widely and specifically expressed in the developing nervous system, with highest concentration at embryonic day 13 in the dorsal root ganglia and ventral spinal cord. At postnatal day 7 Rac3 appears particularly abundant in populations of projection neurons in several regions of the brain, including the fifth layer of the cortex and the CA1-CA3 region of the hippocampus. We generated mice deleted for the Rac3 gene with the aim of analyzing the function of this GTPase in vivo. Rac3 knockout animals survive embryogenesis and show no obvious developmental defects. Interestingly, specific behavioral differences were detected in the Rac3-deficient animals, since motor coordination and motor learning on the rotarod was superior to that of their wild-type littermates. No obvious histological or immunohistological differences were observed at major sites of Rac3 expression. Our results indicate that, in vivo, Rac3 activity is not strictly required for normal development in utero but may be relevant to later events in the development of a functional nervous system.

Normal levels of Rac1 are important for dendritic but not axonal development in hippocampal neurons.

BACKGROUND INFORMATION: Rho family GTPases are required for cytoskeletal reorganization and are considered important for the maturation of neurons. Among these proteins, Rac1 is known to play a crucial role in the regulation of actin dynamics, and a number of studies indicate the involvement of this protein in different steps of vertebrate neuronal maturation. There are two distinct Rac proteins expressed in neurons, namely the ubiquitous Rac1 and the neuron-specific Rac3. The specific functions of each of these GTPases during early neuronal development are largely unknown. RESULTS: The combination of the knockout of Rac3 with Rac1 down-regulation by siRNA (small interfering RNA) has been used to show that down-regulation of Rac1 affects dendritic development in mouse hippocampal neurons, without affecting axons. F-actin levels are strongly decreased in neuronal growth cones following down-regulation of Rac1, and time-lapse analysis indicated that the reduction of Rac1 levels decreases growth-cone dynamics. CONCLUSIONS: These results show that normal levels of endogenous Rac1 activity are critical for early dendritic development, whereas dendritic outgrowth is not affected in hippocampal neurons from Rac3-null mice. On the other hand, early axonal development appears normal after Rac1 down-regulation. Our findings also suggest that the initial establishment of neuronal polarity is not affected by Rac1 down-regulation.

Protein kinase A-mediated synapsin I phosphorylation is a central modulator of Ca2+-dependent synaptic activity.

Protein kinase A (PKA) modulates several steps of synaptic transmission. However, the identification of the mediators of these effects is as yet incomplete. Synapsins are synaptic vesicle (SV)-associated phosphoproteins that represent the major presynaptic targets of PKA. We show that, in hippocampal neurons, cAMP-dependent pathways affect SV exocytosis and that this effect is primarily brought about through synapsin I phosphorylation. Phosphorylation by PKA, by promoting dissociation of synapsin I from SVs, enhances the rate of SV exocytosis on stimulation. This effect becomes relevant when neurons are challenged with sustained stimulation, because it appears to counteract synaptic depression and accelerate recovery from depression by fostering the supply of SVs from the reserve pool to the readily releasable pool. In contrast, synapsin phosphorylation appears to be dispensable for the effects of cAMP on the frequency and amplitude of spontaneous synaptic currents and on the amplitude of evoked synaptic currents. The modulation of depolarization-evoked SV exocytosis by PKA phosphorylation of synapsin I is primarily caused by calmodulin (CaM)-dependent activation of cAMP pathways rather than by direct activation of CaM kinases. These data define a hierarchical crosstalk between cAMP- and CaM-dependent cascades and point to synapsin as a major effector of PKA in the modulation of activity-dependent SV exocytosis.

ADP-ribosylation factor 6 and a functional PIX/p95-APP1 complex are required for Rac1B-mediated neurite outgrowth.

The mechanisms coordinating adhesion, actin organization, and membrane traffic during growth cone migration are poorly understood. Neuritogenesis and branching from retinal neurons are regulated by the Rac1B/Rac3 GTPase. We have identified a functional connection between ADP-ribosylation factor (Arf) 6 and p95-APP1 during the regulation of Rac1B-mediated neuritogenesis. P95-APP1 is an ADP-ribosylation factor GTPase-activating protein (ArfGAP) of the GIT family expressed in the developing nervous system. We show that Arf6 has a predominant role in neurite extension compared with Arf1 and Arf5. Cotransfection experiments indicate a specific and cooperative potentiation of neurite extension by Arf6 and the carboxy-terminal portion of p95-APP1. Localization studies in neurons expressing different p95-derived constructs show a codistribution of p95-APP1 with Arf6, but not Arf1. Moreover, p95-APP1-derived proteins with a mutated or deleted ArfGAP domain prevent Rac1B-induced neuritogenesis, leading to PIX-mediated accumulation at large Rab11-positive endocytic vesicles. Our data support a role of p95-APP1 as a specific regulator of Arf6 in the control of membrane trafficking during neuritogenesis.

betaPIX controls cell motility and neurite extension by regulating the distribution of GIT1.

Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. GIT1/p95-APP1 is a member of a family of GTPase-activating proteins for ARF GTPases that affect endocytosis, adhesion and migration. GIT1 associates with paxillin and a complex including the Rac/Cdc42 exchanging factors PIX/Cool and the kinase PAK. In this study, we show that overexpression of betaPIX induces the accumulation of endogenous and overexpressed GIT1 at large structures similar to those induced by an ArfGAP-defective mutant of GIT1 (p95-C2). Immunohistochemical analysis and immunoelectron microscopy reveal that these structures include the endogenous transferrin receptor. Time-lapse analysis during motogenic stimuli shows that the formation and perinuclear accumulation of the p95-C2-positive structures is paralleled by inhibition of lamellipodium formation and cell retraction. Both dimerization and a functional SH3 domain of betaPIX are required for the accumulation of GIT1 in fibroblasts, which is prevented by the monomeric PIX-PG-DeltaLZ. This mutant also prevents the formation of endocytic aggregates and inhibition of neurite outgrowth in retinal neurons expressing p95-C2. Our results indicate that betaPIX is an important regulator of the subcellular distribution of GIT1, and suggest that alteration in the level of expression of the complex affects the endocytic compartment and cell motility.