The role of microtubules in initial neuronal polarization

Neurons are highly polarized cells with two structurally and functionally distinct compartments, axons and dendrites. This dichotomy is the basis for unidirectional signal propagation, the quintessential function of neurons. During neuronal development, the formation of the axon is the initial step in breaking cellular symmetry and the establishment of neuronal polarity. Although a number of polarity regulators involved in this process have been identified, our understanding of the intracellular mechanisms underlying neuronal polarization still remains fragmentary. In my studies, I addressed the role of microtubule dynamics in initial neuronal polarization. To this end I aimed to investigate the following issues: 1) How do microtubule dynamics and stability change during initial neuronal development? 2) Do microtubules play an instructive role in axon formation? 3) What are possible regulators mediating changes in microtubule dynamics during axon formation? Using hippocampal neurons in culture as a model system for neuronal polarization I first addressed the dynamics of microtubules in early developmental stages of neurons. Assessing posttranslational modifications of tubulin which serve as markers of microtubule turnover I found that microtubule stability is increased in a single neurite already before axon formation and in the axon of morphologically polarized cells. This polarized distribution of microtubule stability was confirmed by testing the resistance of neuronal microtubules to pharmacologically induced depolymerization. The axon of polarized neurons and a single neurite in morphologically unpolarized cells showed increased microtubule stability. Thus, I established a correlation between the identity of a process and its microtubule stability. By manipulating specific regulators of neuronal polarity, SAD kinases and GSK-3beta, I analyzed a possible relation between a polarization of microtubule stability and neuronal polarity. I found that a loss of polarity correlated with a loss of polarized microtubule stability in neurons defective for SAD A and SAD B kinases. In marked contrast, the formation of multiple axons, induced by the inhibition of GSK-3beta, was associated with increased microtubule stability in these supernumerary axons. These results suggested that SAD kinases and GSK-3beta regulate neuronal polarization –at least in part– by modulating microtubule dynamics. To establish a possible causal relation between microtubule dynamics and axon formation I assessed the effects of specific pharmacological alterations of microtubule dynamics on neuronal polarization. I found that application of low doses of the microtubule destabilizing drug nocodazole selectively reduced the formation of future dendrites. Conversely, low doses of the microtubule stabilizing drug taxol led to the formation of multiple axons. I also studied microtubule dynamics in living neurons transfected with GFP-tagged EB3, a protein binding specifically to polymerizing microtubule plus ends. In line with my previous observations I found that microtubules are stabilized along the shaft of the growing axons while dynamic microtubules enrich at the tip of the growing process, suggesting that a well- balanced shift of microtubule dynamics towards more stable microtubules is necessary to induce axon formation. By uncaging a photoactivatable analog of taxol I induced a local stabilization of microtubules at the neurite tip of an unpolarized neuron which was sufficient to favor the site of axon formation. This indicates that a transient stabilization of microtubules is sufficient to trigger axon formation. In summary, my data allow the following conclusions: 1) Microtubule stability correlates with the identity of a neuronal process. 2) Microtubule stabilization causes axon formation. 3) Microtubule stabilization precedes axon formation. I therefore deduce that microtubules are actively involved in the process of axon formation and that local microtubule stabilization in one neuronal process is a physiological signal specifying neuronal polarization