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.