14 January 2005. Light and gravity ensure that tree trunks grow straight up while their roots burrow deep underground. What about neurons, which can project up, down, and anywhere in between? What keeps the axon a pole apart from the dendrites? In this week’s Cell, reports from two independent labs arrive at the conclusion that cellular polarity in neurons is achieved, at least partly, by the differential activation of glycogen synthase kinase 3β (GSK3β). This enzyme has been implicated in Alzheimer disease (AD) because it is one of the kinases that phosphorylates tau, the major constituent of neurofibrillary tangles. GSK3β has also been found to be elevated in AD brain tissue (see Leroy et al., 2002).
The link between tau and neuronal polarity may not be serendipitous. Kozo Kaibuchi and colleagues at Nagoya University, Japan, had previously shown that a protein called CRMP-2 (collapsing response mediator protein-2) is essential for regulating axon growth and that it promotes assembly of microtubules (see also Cole et al., 2004). Tau, of course, is a microtubule binding protein. Now, Kaibuchi, first author Takeshi Yoshimura, and colleagues show that GSK3β phosphorylates not only tau but also CRMP-2.
Previous work had shown that the phosphorylation of threonine 514 (Thr514) on CRMP-2 is crucial for controlling its activity, and that this amino acid lies in a GSK3β consensus phosphorylation site. In the present paper, Yoshimura and colleagues showed that coexpressing CRMP-2 and the GSK3β in COS7 cells causes the mediator protein to be phosphorylated on Thr514. They then wondered how this posttranslational modification might affect axonal growth.
Axonal growth seems a bit of a haphazard affair. In culture, neurons extend tiny growth processes in all directions. After about 12-24 hours, one of these processes rapidly extends to form an axon, and in response, the remaining processes take on the features of dendrites. To see how CRMP-2 might affect this process, the authors used fluorescent antibodies to visually track its location in cultured hippocampal neurons. They found that although total CRMP-2 was elevated in axons sprouting from these neurons, levels of the Thr514 phosphorylated form were 40 percent lower in the vicinity of the growth cone as compared to the shaft. The data suggest that the very tip of the axon contains a pool of unphosphorylated CRMP-2.
Could this spatial regulation of phosphorylation explain the difference between growth of axons vs. dendrites? For one thing, the authors showed that phosphorylation of CRMP-2 inhibits its interaction with tubulin, an essential component of the microtubules that are necessary to elongate an axon. For another, when they mutated Thr514 to remove the phosphorylation site, CRMP-2 became much better at stimulating axon outgrowth than was either the wild-type or a permanently phosphorylated mimic. This, too, points to GSK3β-mediated phosphorylation of CRMP-2 as a crucial factor in preventing axon growth.
Indeed, previous reports have demonstrated that inhibition of GSK3β results in enhanced neurite outgrowth (see Munoz-Montano et al.. 1999), and the authors confirmed this using several inhibitors of the kinase, including lithium chloride (see also ARF recent news story on how lithium may reduce production of amyloid-β by inhibiting GSK3α). When the authors added these inhibitors to cultured hippocampal neurons, they found that the number of neurons with tau-1 positive axons increased by up to twofold. They obtained a similar effect using RNAi to knock down GSK3β, whereas expression of a constitutively active kinase (which would phosphorylate CRMP-2) cut the number of neurons with tau-1 positive axons almost in half.
Next, the authors turned to events upstream of GSK3β. They questioned the role of NT-3 and BDNF, which have been shown to inhibit GSK3β in neurons and enhance axonal elongation and branching. Could these cytokines evoke longer axons because they reduce levels of phosphorylated CRMP-2?
To test this, the investigators added NT-3 to hippocampal neurons. They found that levels of phosphorylated CRMP-2 decreased by almost 40 percent in the growth cone, less in the axonal shafts, and not at all in the cell body, supporting the idea that a spatial gradient of phosphorylated CRMP-2 contributes to cell polarity.
All told, the work supports a scenario whereby inactivation of GSK3β by phosphorylation, possibly in response to growth factor signaling, leads to relatively higher levels of unphosphorylated CRMP-2 at axonal growth cones (see diagram). And indeed, in the second paper, Yi Rao and colleagues at the Shanghai Institute of Biological Sciences and the National Institute of Biological Sciences in Beijing, substantially backed up these findings, and then added some.
Model for axon growth
Active, unphosphorylated CRMP-2 binds to tubulin heterodimers to promote microtubule assembly. GSK3β can block this by phosphorylating CRMP-2. Signaling events that activate PI3-kinase may stimulate axon growth by activating Akt, which in turn can inactivate GSK3β. [Image courtesy Kozo Kaibuchi.]
Rao’s group found that GSK3β is spatially regulated. In a set of experiments similar to those of the Kaibuchi group on CRMP-2, first author Hui Jiang and colleagues showed that the ratio of inactive GSK3β (phosphorylated at serine 9) to active, unphosphorylated kinase was highest in the tips of the axon. This would explain Kaibuchi’s findings that relatively higher levels of unphosphorylated CRMP-2 (which, remember, is phosphorylated by GSK3β) is driving axon development, and hence, neural polarity.
Moreover, Jiang and colleagues noticed that GSK3β made constitutively active by way of mutation at position 9 led to a decrease in axon formation; half the cultured neurons failed to form axons if they expressed this active kinase, as opposed to only eight percent failure in control cultures. The opposite experiment, inhibiting GSK3β, led to multiple axons. What’s more, these additional axons apparently were functional. Use of the small dye FM4-64 revealed that the axons recycled synaptic vesicles after stimulation with potassium, a sign that they are capable of supporting release of neurotransmitters.
As for upstream signaling events, Jiang found that the kinase Akt also contributes to polarity, again tying in with the findings of Kaibuchi’s group (see, for example, ARF related news story on how Akt may protect neurons in animal models of amyotrophic lateral sclerosis). When Jiang treated cultured hippocampal neurons with LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3K), he found that not only was phosphorylation of Akt (on serine 473) completely inhibited, but so too was the phosphorylation of GSK3β at serine 9. The authors also found that a constitutively active Akt led to the formation of multiple axons, which would make sense as it would inactivate GSK3β and so lead to more unphosphorylated CRMP-2. As activation of Akt by PI3K can be driven by NT-3/BDNF signaling, this also fits well with the findings of Kaibuchi’s group (see diagram).
While painting a fairly detailed picture of events that impinge on neuronal polarity, these two papers also raise many questions. While “GSK3β activity is of central importance,” as Rao and colleagues write, the complete set of figures remains nebulous for now. What other factors lie downstream of GSK3β—perhaps tau? What about upstream and sidestream? Yoshimura and colleagues showed that GSK3β alone is insufficient for phosphorylation of CRMP-2 in vitro. Other factors are in the mix—perhaps Cdk5, which can phosphorylate CRMP-2 at serines 518 and 522, may be involved? Rao’s group also notes that none of this data explains how Par3/Par6, APC, CDC42 and Rap1B fit in, all of which are known to affect neuronal polarity.
And how might all of this play out in AD or other neurodegenerative diseases? The role of GSK3β in tau hyperphosphorylation is well described, but large gaps remain. “These observations raise the possibility that hyperphosphorylation of CRMP-2 is involved in the development of neurofibrillary tangles and plaque neurites,” suggest Yoshimura and colleagues. This signaling pathway also offers an additional explanation for the neuroprotective effect of BDNF, and suggests that GSK3β inhibitors, in addition to possibly preventing hyperphosphorylation of tau, might also stimulate axonal regeneration.—Tom Fagan.
Yoshimura T, Kawano Y, Arimura N, Kawabata S, Kikuchi A, Kaibuchi K. GSK-3β regulates phosphorylation of CRMP-2 and neuronal polarity. Cell. 14 January 2005;120:137-149. Abstract
Jiang H, Guo W, Liang X, Rao Y. Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK-3β and its upstream regulators. Cell. 14 January 2005;120:123-135. Abstract