Glycogen synthase kinase 3beta and the regulation of axon growth.

One of the earliest hallmarks that distinguish growing axons from dendrites is their growth rate; axons grow faster than dendrites. In vertebrates, where axons are required to grow for considerable distances, particularly in the peripheral nervous system, a fast axon growth rate is a requisite property. In neurons that respond to the neurotrophin growth factor/nerve growth factor with increased axon growth rates, two distinct intracellular signalling pathways are recruited: the MAPK (mitogen-activated protein kinase) pathway and the phosphatidylinositol-3 kinase pathway. The activation of either pathway leads to changes in microtubule dynamics within growing axons and growth cones and these underlie fast axon growth rates. Microtubule dynamics is regulated by microtubule-associated proteins and in the MAPK pathway this function is subserved by microtubule-associated protein 1B, whereas in the phosphatidylinositol-3 kinase pathway, adenomatous polyposis coli is the regulating microtubule-associated protein.

Microtubule-associated protein 1B phosphorylation by glycogen synthase kinase 3beta is induced during PC12 cell differentiation.

In recent studies we have demonstrated that glycogen synthase kinase 3beta (GSK3beta) and its substrate microtubule-associated protein 1B (MAP1B) regulate the microtubule cytoskeleton during axon outgrowth. To further examine the role GSK3beta plays in axon outgrowth we investigated the expression of GSK3beta and its activity towards MAP1B during nerve growth factor (NGF)-stimulated PC12 cell differentiation. Levels of GSK3beta expression increase relatively little during the course of differentiation. However, the expression of a novel GSK3beta isoform characterised by a reduced mobility on SDS gels is induced by NGF. Expression of this isoform and the GSK3beta-phosphorylated isoform of MAP1B (MAP1B-P) are induced in parallel in response to NGF. This increase lags behind initial neurite formation and the expression of MAP1B in these cells by about two days and coincides with a period when the majority of cells are extending existing neurites. MAP1B and GSK3beta are expressed throughout the PC12 cell but MAP1B-P expression is restricted to the growth cones and neurites. Consistent with these observations, we find that neurite extension is more sensitive to the GSK3 inhibitor Li+ than neurite formation and that this correlates with an inhibition of MAP1B phosphorylation. Additionally, GSK3beta from PC12 cells not exposed to NGF can not phosphorylate MAP1B in vitro. However, a soluble factor in differentiated PC12 cell extracts depleted of GSK3beta can activate MAP1B phosphorylation from undifferentiated cell extracts otherwise devoid of kinase activity. These experiments provide evidence for an NGF-mediated regulation of MAP1B phosphorylation in growing neurites by the induction of a novel isoform of GSK3beta.

Glycogen synthase kinase 3beta phosphorylation of microtubule-associated protein 1B regulates the stability of microtubules in growth cones.

We have recently shown that glycogen synthase kinase 3beta (GSK3beta) phosphorylates the microtubule-associated protein (MAP) 1B in an in vitro kinase assay and in cultured cerebellar granule cells. Mapping studies identified a region of MAP1B high in serine-proline motifs that is phosphorylated by GSK3beta. Here we show that COS cells, transiently transfected with both MAP1B and GSK3beta, express high levels of the phosphorylated isoform of MAP1B (MAP1B-P) generated by GSK3beta. To investigate effects of MAP1B-P on microtubule dynamics, double transfected cells were labelled with antibodies to tyrosinated and detyrosinated tubulin markers for stable and unstable microtubules. This showed that high levels of MAP1B-P expression are associated with the loss of a population of detyrosinated microtubules in these cells. Transfection with MAP1B protected microtubules in COS cells against nocodazole depolymerisation, confirming previous studies. However, this protective effect was greatly reduced in cells containing high levels of MAP1B-P following transfection with both MAP1B and GSK3beta. Since we also found that MAP1B binds to tyrosinated, but not to detyrosinated, microtubules in transfected cells, we propose that MAP1B-P prevents tubulin detyrosination and subsequent conversion of unstable to stable microtubules and that this involves binding of MAP1B-P to unstable microtubules. The highest levels of MAP1B-P are found in neuronal growth cones and therefore our findings suggest that a primary role of MAP1B-P in growing axons may be to maintain growth cone microtubules in a dynamically unstable state, a known requirement of growth cone microtubules during pathfinding. To test this prediction, we reduced the levels of MAP1B-P in neuronal growth cones of dorsal root ganglion cells in culture by inhibiting GSK3beta with lithium. In confirmation of the proposed role of MAP1B-P in maintaining microtubule dynamics we found that lithium treatment dramatically increased the numbers of stable (detyrosinated) microtubules in the growth cones of these neurons.

Inhibition of GSK-3beta leading to the loss of phosphorylated MAP-1B is an early event in axonal remodelling induced by WNT-7a or lithium.

WNT-7a induces axonal spreading and branching in developing cerebellar granule neurons. This effect is mediated through the inhibition of GSK-3beta, a serine/threonine kinase and a component of the WNT pathway. Lithium, an inhibitor of GSK-3beta, mimics WNT-7a in granule cells. Here we examined further the effect of GSK-3beta inhibition on cytoskeletal re-organisation. Lithium induces axonal spreading and increases growth cone area and perimeter. This effect is associated with the absence or reduction of stable microtubules in spread areas. Lithium induces the loss of a phosphorylated form of MAP-1B, a microtubule associated protein involved in axonal outgrowth. Down-regulation of the phosphorylated MAP-1B, MAP-1B-P, from axonal processes occurs before axonal remodelling is evident. In vitro phosphorylation assays show that MAP-1B-P is generated by direct phosphorylation of MAP-1B by GSK-3beta. WNT-7a, like lithium, also leads to loss of MAP-1B-P from spread axons and growth cones. Our data suggest that WNT-7a and lithium induce changes in microtubule dynamics by inhibiting GSK-3beta which in turn lead to changes in the phosphorylation of MAP-1B. These findings suggest a novel role for GSK-3beta and WNTs in axonal remodelling and identify MAP-1B as a new target for GSK-3beta and WNT.

Localisation of microtubule-associated protein 1B phosphorylation sites recognised by monoclonal antibody SMI-31.

MAP 1B is a microtubule-associated phosphoprotein that is expressed early in neurons and plays a role in axon growth. MAP 1B has two types of phospho-isoforms, one of which is developmentally down-regulated after neuronal maturation and one of which persists into adulthood. Because phosphorylation regulates MAP 1B binding activity, characterisation of the phosphorylation sites and identification of the corresponding kinases/phosphatases are important goals. We have characterised the developmentally down-regulated phosphorylation sites recognised by monoclonal antibody (mAb) SMI-31. We purified MAP 1B from neonatal rat brain and mapped the mAb SMI-31 sites to specific MAP 1B fragments after chemical cleavage. We then developed an in vitro kinase assay by using a high-speed spin supernatant from neonatal rat brain in the presence of ATP and recombinant proteins encoding selective regions of the MAP 1B molecule. Phosphorylation of the recombinant protein was detected on western blots using mAb SMI-31. This analysis showed that mAb SMI-31 recognises two recombinant proteins corresponding to residues 1,109-1,360 and 1,836-2,076 of rat MAP 1B after in vitro phosphorylation. The former phosphorylation site was further defined in the in vitro kinase assay by inhibition with peptides and antibodies from candidate regions of the MAP 1B sequence. This approach identified a region of 20 amino acids, from 1,244 to 1,264, characterised by a high concentration of serines immediately upstream of prolines, indicating that the kinase responsible is a proline-directed serine kinase.

The neurofilament antibody RT97 recognises a developmentally regulated phosphorylation epitope on microtubule-associated protein 1B.

Microtubules are important for the growth and maintenance of stable neuronal processes and their organisation is controlled partly by microtubule-associated proteins (MAPs). MAP 1B is the first MAP to be expressed in neurons and plays an important role in neurite outgrowth. MAP 1B is phosphorylated at multiple sites and it is believed that the function of the protein is regulated by its phosphorylation state. We have shown that the monoclonal antibody (mAb) RT97, which recognises phosphorylated epitopes on neurofilament proteins, fetal tau, and on Alzheimer's paired helical filament-tau, also recognises a developmentally regulated phosphorylation epitope on MAP 1B. In the rat cerebellum, Western blot analysis shows that mAb RT97 recognises the upper band of the MAP 1B doublet and that the amount of this epitope peaks very early postnatally and decreases with increasing age so that it is absent in the adult, despite the continued expression of MAP 1B in the adult. We confirmed that mAb RT97 binds to MAP 1B by showing that it recognises MAP 1B immunoprecipitated from postnatal rat cerebellum using polyclonal antibodies to recombinant MAP 1B proteins. We established that the RT97 epitope on MAP 1B is phosphorylated by showing that antibody binding was abolished by alkaline phosphatase treatment of immunoblots. Epitope mapping experiments suggest that the mAb RT97 site on MAP 1B is near the N-terminus of the molecule. Despite our immunoblotting data, immunostaining of sections of postnatal rat cerebellum with mAb RT97 shows a staining pattern typical of neurofilaments with no apparent staining of MAP 1B. For instance, basket cell axons and axons in the granule cell layer and white matter stained, whereas parallel fibres did not. These results suggest that the MAP 1B epitope is masked or lost under the immunocytochemical conditions in which the cerebellar sections are prepared. The upper band of the MAP 1B doublet is believed to be predominantly phosphorylated by proline-directed protein kinases (PDPKs). PDPKs are also good candidates for phosphorylating neurofilament proteins and tau and therefore we postulate that the sites recognised by RT97 on these neuronal cytoskeletal proteins may be phosphorylated by similar kinases. Important goals are to determine the precise location of the RT97 epitope on MAP 1B and the kinase responsible.

An analysis of an axonal gradient of phosphorylated MAP 1B in cultured rat sensory neurons.

The present study investigated the cellular distribution of a developmentally regulated phosphorylated form of MAP 1B recognized by monoclonal antibody (mAb) 150 in cultures of dorsal root ganglia. The cell soma and the whole axon, when it first appears, are labelled, but longer axons label with a proximodistal gradient, such that the cell soma and proximal axon become unlabelled, whilst the distal axon and growth cone label strongly. Double-labelling experiments with mAb 150 and a polyclonal antibody (N1-15) that recognizes all forms of MAP 1B demonstrated that MAP 1B is distributed along the entire length of axons with gradients, so the gradient of phosphorylated MAP 1B is not due to a loss or absence of MAP 1B from the proximal axon. The proportion of axons from 20 h cultures that were labelled with a mAb 150 gradient was at least 80% and this proportion was independent of the nerve growth factor concentration of the culture medium. Analysis of axons ranging in length from 100 to 700 microm and labelled with a gradient showed that the unlabelled proximal portions of axons increased in length more slowly than the labelled distal axon. Axons labelled along their entire length accounted for no more than 19% of th axonal population and analysis of these showed them to be frequently < 400 microm long. After simultaneously fixing and detergent-extracting cultures this proportion rose significantly to 93%, suggesting that in the proximal axon the mAb 150 epitope is masked by some factor(s) that is removed by detergent extraction. The possibility that mAb 150 could not access the epitope in the proximal axon was discounted because another IgM, mAb 125, which recognizes a different phosphorylation epitope on MAP 1B, labelled the proximal axon of conventionally fixed cultures. In growth cones of fixed and extracted neurons examined by immunofluorescence, the mAb 150 labelling strongly colocalized to bundled microtubules in the distal axon shaft and the C-domain. In the P-domain, mAb 150 staining was weaker and more widely distributed than the microtubules. Immunogold electron microscopy confirmed that antibody N1-15 and mAb 150 strongly labelled the bundled microtubules in the C-domain and also showed that individual microtubules in the P-domain, some of which lie alongside actin filament bundles of filopodia, were labelled lightly and discontinuously with both antibodies. This suggests that the phosphorylated isoform of MAP 1B recognized by mAb 150 may be microtubules and actin filaments in the P-domain.