Cytoskeleton dynamics and axon behaviour: a role for Wnt signalling

The formation of a functional brain requires the assembly of appropriate nerve connections. During development, nerve cells (neurons) extend long processes called axons that grow extensively in search for their target tissue. Along their trajectory axons encounter a number of signals that elicit different responses such as attraction, pausing or repulsion. The tip of the axon or growth cone is particularly sensitive to these signals. For example, certain signals induce pausing, extension, and turning. These different responses are accomplished by the ability of these extracellular cues to 'talk' to and modify the skeleton (cytoskeleton) inside neurons. This cytoskeleton is formed by dynamic polymers that change in length, organization and direction of growth allowing nerve cells to respond quickly to changes in the environment. Although great progress has been made in the identification of large number of axon guidance cues, little is known about the mechanisms by which these extracellular cues modulate the behaviour of axons and particularly how these signals change the cytoskeleton in axons. Our laboratory has been studying the function of Wnts, molecules released by synaptic targets, in the formation of nerve connections. Wnts induce profound changes in the behaviour of axons as axons pause, form branches and change their shape when they encounter Wnt proteins. Following these changes, Wnts stimulate the formation of synapses by bringing components necessary for the release of neurotransmitters. This grant proposal addresses the mechanisms by which Wnt molecules change the behaviour of axons and the relationship with changes in the cytoskeleton. As we mainly focus on regenerating axons, our studies will new shed light into possible therapeutic strategies that can be used for nerve repair after injury.

The formation of a functional nervous system requires the establishment of appropriate neuronal connections during development. Axons navigate through a complex environment in search for their appropriate synaptic targets. Along their trajectory, axons encounter a number of guidance cues that control the direction of axonal growth and behavior. In addition, target-derived factors modulate the terminal axon arborization, a process that resides and likely to modulate the assembly the synapses. These signals induce different behaviors, from axon outgrowth and turning to growth cone pausing and remodeling. Changes in the neuronal cytoskeleton are obligatory to induce these cellular responses, but the precise changes elicited by axon guidance cues on the cytoskeleton remain poorly understood. Wnt proteins have been shown to regulate axon guidance, dendritogenesis, axon arborisation and synapse formation. Our group has been studying the function of Wnts in neuronal connectivity, in particular their function as target-derived signals that modulate the terminal remodelling of axons. We have shown that Wnts modulate the both dynamics and organization of the microtubule and actin cytoskeletons in developing and regenerating axons. This central aim of this proposal is to elucidate the mechanisms by which Wnts regulate axon behaviour by focusing on the cytoskeleton. Using time-lapse fluorescence microscopy and fluorescent-tagged cytoskeletal molecules, we will dissect the sequence of events leading to remodelling. Our initial screen has identified key target molecules regulated by Wnts. These cytoskeletal-associated proteins could mediate Wnt induced axon remodelling. We will interfere with their function by expressing dominant negative and/or constitute active mutants in Wnt responsive axons. Finally, we will examine Wnt signalling deficient mice for possible defects in axon pathfinding. Our studies will shed new light into the mechanisms that modulate the neuronal cytoskelet