It is widely believed that signature patterns of microtubule polarity orientation within axons and dendrites underlie compositional and morphological differences that distinguish these neuronal processes from one another. Axons of vertebrate neurons display uniformly plus-end-distal microtubules, whereas their dendrites display non-uniformly oriented microtubules. Fly axons also display uniformly plus-end-distal microtubules, but their dendritic microtubules are nearly uniformly minus-end-distal. Discussed in this article are the history of these findings, their implications for the regulation of neuronal polarity across the animal kingdom, and potential mechanisms by which neurons establish the distinct microtubule polarity patterns that define axons and dendrites.

MAP1B is a developmentally regulated microtubule-associated phosphoprotein that regulates microtubule dynamics in growing axons and growth cones. We used mass spectrometry to map 28 phosphorylation sites on MAP1B, and selected for further study a putative primed GSK3β site and compared it with two nonprimed GSK3β sites that we had previously characterised. We raised a panel of phosphospecific antibodies to these sites on MAP1B and used it to assess the distribution of phosphorylated MAP1B in the developing nervous system. This showed that the nonprimed sites are restricted to growing axons, whereas the primed sites are also expressed in the neuronal cell body. To identify kinases phosphorylating MAP1B, we added kinase inhibitors to cultured embryonic cortical neurons and monitored MAP1B phosphorylation with our panel of phosphospecific antibodies. These experiments identified dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK1A) as the kinase that primes sites of GSK3β phosphorylation in MAP1B, and we confirmed this by knocking down DYRK1A in cultured embryonic cortical neurons by using shRNA. DYRK1A knockdown compromised neuritogenesis and was associated with alterations in microtubule stability. These experiments demonstrate that MAP1B has DYRK1A-primed and nonprimed GSK3β sites that are involved in the regulation of microtubule stability in growing axons.

Microtubules are nearly uniformly oriented in the axons of vertebrate neurons, but are non-uniformly oriented in their dendrites. Studies to date suggest a scenario for establishing these microtubule patterns whereby microtubules are transported into the axon and nascent dendrites with plus-ends-leading, and then additional microtubules of the opposite orientation are transported into the developing dendrites. Here, we used contemporary tools to confirm that depletion of kinesin-6 (also called CHO1/MKLP1 or kif23) from rat sympathetic neurons causes a reduction in the appearance of minus-end-distal microtubules in developing dendrites, which in turn causes them to assume an axon-like morphology. Interestingly, we observed a similar phenomenon when we depleted kinesin-12 (also called kif15 or HKLP2). Both motors are best known for their participation in mitosis in other cell types, and both are enriched in the cell body and dendrites of neurons. Unlike kinesin-12, which is present throughout the neuron, kinesin-6 is barely detectable in the axon. Accordingly, depletion of kinesin-6, unlike depletion of kinesin-12, has no impact on axonal branching or navigation. Interestingly, depletion of either motor results in faster growing axons with greater numbers of mobile microtubules. Based on these observations, we posit a model whereby these two motors generate forces that attenuate the transport of microtubules with plus-ends-leading from the cell body into the axon. Some of these microtubules are not only prevented from moving into the axon, but are driven with minus-ends-leading into developing dendrites. In this fashion, these so-called “mitotic” motors co-regulate the microtubule patterns of axons and dendrites.

Developing neurons express a motor protein called kinesin-5 (also called kif11 or Eg5) which acts as a 'brake' on the advance of the microtubule array during axonal growth. Pharmacological inhibition of kinesin-5 causes the developing axon to grow at a faster rate, retract less and grow past cues that would otherwise cause it to turn. Here we demonstrate that kinesin-5 is also expressed in adult neurons, albeit at lower levels than during development. We hypothesized that inhibiting kinesin-5 might enable adult axons to regenerate better and to overcome repulsive molecules associated with injury. Using adult mouse dorsal root ganglion neurons, we found that antikinesin-5 drugs cause axons to grow faster and to cross with higher frequency onto inhibitory chondroitin sulfate proteoglycans. These effects may be due in part to changes in the efficiency of microtubule transport along the axonal shaft as well as enhanced microtubule entry into the distal tip of the axon. Effects observed with the drugs are further enhanced in some cases when they are used in combination with other treatments known to enhance axonal regeneration. Collectively, these results indicate that anti-kinesin-5 drugs may be a useful addition to the arsenal of tools used to treat nerve injury.

In order for growth cones to turn, microtubules from the central domain must preferentially invade the peripheral domain in the direction of the turn. Recent studies suggest that kinesin-5 (also called Eg5 or kif11) suppresses the invasion of microtubules into the peripheral domain on the side of the growth cone opposite of the direction of turning. In theory, kinesin-5 could elicit these effects by acting on the microtubules within the peripheral domain itself, or by acting on microtubules in the central domain or in the transition zone between these two domains. In rat neurons expressing kinesin-5-EGFP, we documented the presence of kinesin-5 in both domains of the growth cone and especially enriched in the transition zone. We then focally inactivated kinesin-5 in various regions of the growth cone, using micro-CALI (chromophore-assisted laser inactivation). We found that a greater invasion of microtubules into the peripheral domain occurred when kinesin-5 was inactivated specifically in the transition zone. However, there was no effect on microtubule invasion into the peripheral domain when kinesin-5 was inactivated in the peripheral domain itself or in the central domain. In other experiments, frog growth cones were observed to turn toward a gradient of a drug that inhibits kinesin-5, confirming that asymmetric inactivation of kinesin-5 can cause the growth cone to turn. Finally, expression of a phospho-mutant of kinesin-5 resulted in greater microtubule invasion throughout the peripheral domain and an inhibition of growth cone turning, implicating phosphorylation as a means by which kinesin-5 is regulated in the growth cone.