During the past decade enormous advances have been made in our understanding of the basic molecular machinery that is involved in the development of neuronal polarity. Far from being mere structural elements, microtubules are emerging as key determinants of neuronal polarity. Here we review the current understanding of the regulation of microtubule assembly, organization and dynamics in axons and dendrites. These studies provide new insight into microtubules' function in neuronal development and their potential contribution to plasticity.

Neurons in culture can have fundamentally distinct morphologies which permit their cytological identification and the recognition of their neurites as axons or dendrites. Microtubules may have a role in determining morphology by the selective stabilization of spatially distinct microtubule subsets. The plasticity of a neurite correlates inversely with the stability of its component microtubules: microtubules in growth cones are very dynamic, and in initial neurites there is continuous incorporation of labelled subunits, whereas in mature neurites, microtubules are highly stabilized. The binding of microtubule-associated proteins to the microtubules very probably contributes to this stability. Cerebellar neurons in dissociated culture initially extend exploratory neurites and, after a relatively constant interval, become polarized. Polarity becomes evident when a single neurite exceeds the others in length. These stable neurites cease to undergo the retractions and extensions characteristic of initial neurites and assume many features of axons and dendrites. We have now studied the role of the neuronal microtubule-associate protein tau in neurite polarization by selectively inhibiting tau expression by the addition of antisense oligonucleotides to the culture media. Although the extension of initial exploratory neurites occurred normally, neurite asymmetry was inhibited by the failure to elaborate an axon.

In dissociated-cell cultures prepared from the embryonic rat hippocampus, neurons establish both axons and dendrites, which differ in geometry, in ultrastructure, and in synaptic polarity. We have used immunocytochemistry with monoclonal antibodies to study the regional distribution of beta-tubulin and micro-tubule-associated protein 2 (MAP2) in hippocampal cultures and their localization during early stages of axonal and dendritic development. After development for a week or more in culture, when axons and dendrites were well-differentiated, the distribution of these two proteins was quite different. Beta-tubulin was present throughout the nerve cell, in soma, dendrites, and axon. It was also present in all classes of non-neuronal cells, astrocytes, fibroblasts, and a presumptive glial progenitor cell. In contrast, MAP2 was preferentially localized to nerve cells; within neurons, MAP2 was present in soma and dendrites, but little or no immunostaining was detectable in axons. Both beta-tubulin and MAP2 were present in nerve cells at the time of plating. From the earliest stages of process extension, beta-tubulin was present in all neuronal processes, both axons and dendrites. Surprisingly, MAP2 was also initially present in both axons and dendrites, extending as far as the axonal growth cone. With subsequent development, MAP2 staining was selectively lost from the axon so that after 1 week in vitro little or no axonal staining remained. Taken together with earlier results (Cáceres et al., 1984a), these data indicate that the establishment of neuronal polarity, as manifested by the molecular differentiation of the axonal and dendritic cytoskeleton, occurs largely under endogenous control, even under culture conditions in which cell interactions are greatly restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

In cultured neurons, axon formation is preceded by the appearance in one of the multiple neurites of a large growth cone containing a labile actin network and abundant dynamic microtubules. The invasion-inducing T-lymphoma and metastasis 1 (Tiam1) protein that functions as a guanosine nucleotide exchange factor for Rac1 localizes to this neurite and its growth cone, where it associates with microtubules. Neurons overexpressing Tiam1 extend several axon-like neurites, whereas suppression of Tiam1 prevents axon formation, with most of the cells failing to undergo changes in growth cone size and in cytoskeletal organization typical of prospective axons. Cytochalasin D reverts this effect leading to multiple axon formation and penetration of microtubules within neuritic tips devoid of actin filaments. Taken together, these results suggest that by regulating growth cone actin organization and allowing microtubule invasion within selected growth cones, Tiam1 promotes axon formation and hence participates in neuronal polarization.

In this study we have examined the cellular functions of ERM proteins in developing neurons. The results obtained indicate that there is a high degree of spatial and temporal correlation between the expression and subcellular localization of radixin and moesin with the morphological development of neuritic growth cones. More importantly, we show that double suppression of radixin and moesin, but not of ezrin–radixin or ezrin–moesin, results in reduction of growth cone size, disappearance of radial striations, retraction of the growth cone lamellipodial veil, and disorganization of actin filaments that invade the central region of growth cones where they colocalize with microtubules. Neuritic tips from radixin–moesin suppressed neurons displayed high filopodial protrusive activity; however, its rate of advance is 8–10 times slower than the one of growth cones from control neurons. Radixin–moesin suppressed neurons have short neurites and failed to develop an axon-like neurite, a phenomenon that appears to be directly linked with the alterations in growth cone structure and motility. Taken collectively, our data suggest that by regulating key aspects of growth cone development and maintenance, radixin and moesin modulate neurite formation and the development of neuronal polarity.

Coordinated microtubule and microfilament changes are essential for the morphological development of neurons; however, little is know about the underlying molecular machinery linking these two cytoskeletal systems. Similarly, the indispensable role of RhoGTPase family proteins has been demonstrated, but it is unknown how their activities are specifically regulated in different neurites. In this paper, we show that the cytoplasmic dynein light chain Tctex-1 plays a key role in multiple steps of hippocampal neuron development, including initial neurite sprouting, axon specification, and later dendritic elaboration. The neuritogenic effects elicited by Tctex-1 are independent from its cargo adaptor role for dynein motor transport. Finally, our data suggest that the selective high level of Tctex-1 at the growth cone of growing axons drives fast neurite extension by modulating actin dynamics and also Rac1 activity.

How a neuron becomes polarized remains largely unknown. Results obtained with a function-blocking antibody and an siRNA targeting the insulin-like growth factor-1 (IGF-1) receptor suggest that an essential step in the establishment of hippocampal neuronal polarity and the initiation of axonal outgrowth is the activation of the phosphatidylinositol 3-kinase (PI3k)-Cdc42 pathway by the IGF-1 receptor, but not by the TrkA or TrkB receptors.

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