Inhibition of GSK-3ß induces microtubule unbundling and loss of stable microtubules
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Neuronal networks are generated gradually by a complex pattern of axon guidance, target selection and synapse formation. Although several signalling molecules involved in axon guidance and target selection have been identified, the signals controlling synapse formation remain largely unknown. My laboratory is studying how. We are particularly interested in understanding how signalling molecules of the WNT family regulate the neuronal cytoskeleton during axon guidance and the formation of synapses in the mouse CNS.
In the last few years, my laboratory has examined the function of WNT proteins and some of the downstream components in the formation of neuronal connections. Using in vitro and in vivo approaches we showed that WNT-7a induces axonal remodelling characterised by axonal shortening, enlargement of the growth cone and spreading along the axon. In addition, WNT-7a increases the level of synapsin I, a presynaptic protein at remodelled areas of the axon. These effects are mediated through the inhibition of GSK-3ß, a serine/threonine kinase and a component of the WNT signalling pathway. More importantly, analyses of the Wnt-7a mutant mice showed that WNT-7a as a synaptogenic factor in the mouse cerebellum. These findings have two important implications. Firstly, we have identified WNT-7a as a post-synaptic signal that regulates axonal remodelling and synaptic differentiation of central neurons. Very little is known about how central synapses are formed, in particular, how pre-synaptic differentiation is regulated; therefore, our results provide the first handle on the mechanisms involved in central synaptogenesis. Secondly, it demonstrates for the first time that a deficiency in WNT function affects the formation and maturation of neuronal connections.
Inhibition of GSK-3ß induces microtubule unbundling and loss of stable microtubules
WNT-7a induces axonal spreading and increases synaptic proteins at spread areas
To begin to address how WNT-7a induces axonal remodelling, we have examined the microtubule organisation in remodelled axons. We found that WNT-7a induces re-organisation of microtubules characterised by microtubule unbundling at spread areas and the formation of looped microtubules at terminal growth cones. These changes in microtubule organisation occur concomitantly with the loss of a phosphorylated form of MAP-1B, a microtubule associated protein that control microtubule stability. We found that GSK-3ß directly phosphorylates MAP-1B. Our studies suggest that microtubule remodelling induced by WNT-7a is achieved by inhibition of GSK-3ß resulting in changes in the phosphorylation of MAPs.
To address the mechanisms that control GSK-3ß activity, we have recently focused our studies on the cytoplasmic protein, Dishevelled, an inhibitor of GSK-3ß in the WNT signalling pathway. We are taking cellular, biochemical and transgenic approaches to determine the role of DVL-1 in the regulation of GSK-3ß in developing axons.
Another major project in the lab is focused on understanding the mechanism by which WNTs regulate the clustering and levels of synaptic proteins to the pre-synaptic terminal. Inhibition of the serine/threonine kinase GSK-3ß is involved in this process. We are taking molecular and cellular approaches to understand how the activity of GSK-3ß regulates this process.
Our studies on WNT-7a led to the finding that lithium, a known inhibitor of GSK-3ß and mood stabiliser, induces axonal remodelling and increases synaptic protein levels. These results raise the interesting possibility that modulation of GSK-3ß plays a role in synaptic plasticity and behavioural control. More importantly, the effects of lithium during the treatment of bipolar disorders could be due, in part, to GSK-3ß inhibition. Due to the high toxicity of lithium, there is a great need for developing new drugs for mood regulation. We are currently analysing a number of mood stabilising drugs for their effect on GSK-3ß and neuronal plasticity.
1. Lucas F. R. and Salinas P. C. (1997) Wnt-7A induces axonal remodeling and increases synapsin I levels in cerebellar neurons. Developmental Biology 192: 31-44.
2. Lucas, F. R., Goold, R., Gordon-Weeks, P. and Salinas, P. C. (1998) Inhibition of GSK-3ß leading to the loss of phosphorylated MAP-1B are early events in axonal remodelling induced by WNT-7a or lithium. J. Cell Science. 111: 1351-1361.
3. Salinas P. C. (1998) WNT factors in axonal remodelling and synaptogenesis. Cell Behaviour: Control and Mechanism of Motility (edited by JM Lackie, GA Dunn and GE Jones). Biochemical Society Symposium 65, pp. 101-109. Portland Press, London.
4. Millar S. E., Willert K., Salinas P. C., Nusse R., Sussman D. J. and Barsh G. S. (1999) WNT signaling in the control of hair growth and structure. Developmental Biology 207: 133-149.
5. Hughes S. M. and Salinas P. C. (1999) Intrinsic and extrinsic influences on muscle fibre type: Coordination of muscle fibre and motoneuron diversification. Current Opinion in Neurobiology 9: 54-64.
6. Salinas P. C. and Hall A. (1999) Lithium and Synaptic Plasticity. Bipolar Disorders. 2: 87-90.
7. Meijer L., Thunnissen, A. W.H., White, A., Nikolic M., Tsai L-H., Cleverley K.C., Salinas C. P., Walter J., Garnier M., Wu Y-Z., Biernat J., Mandelkow E-M., Kim S-H., and Pettit G. R. (2000) Inhibition of cyclin-dependent kinases, GSK-3beta and CK1 by hymenialdisine, a marine sponge constituent. Chemistry and Biology. 7: 51-63.
8. Hall A. C., Lucas F. R. and Salinas P. C. (2000) Axonal remodelling and synaptic differentiation in the cerebellum is regulated by WNT-7a signalling. Cell 100: 525-535.