Expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in sprouting and regenerating axons in vitro

We have developed a novel culture system for studying axonal regeneration. Short lengths of spinal nerves with their attached dorsal root ganglia were removed from adult mice, explanted into Matrigel and maintained in serum-free medium for up to eight days. Profuse outgrowth of unfasciculated, naked axons occurred within 6 h from the cut ends of the peripheral nerve, dorsal roots and eventually from the ganglion itself, and continued to grow throughout the observation period. Some axons were entirely smooth, whilst others showed prominent varicosities. The former stained with antibody RT97, a marker for large-calibre, myelinated axons, whilst the latter stained with antibodies to calcitonin gene-related peptide, predominantly a marker for unmyelinated and small-diameter myelinated sensory axons. All axons stained with a monoclonal antibody (150) that recognizes a developmentally regulated phosphorylated isoform of the microtubule-associated protein 1B [Gordon-Weeks P. R. et al. (1993) Eur. J. Neurosci. 5, 1302-1311]. Monoclonal antibody 150 staining was observed along the entire length of all axons growing out of the explant; the proximal regions of these axons within the explant itself did not stain. The staining extended to the growth cones, which had elaborate morphologies. Other antibodies (e.g. to growth-associated protein 43) labelled axons within the nerve, as well as those growing in Matrigel. In preparations where the peripheral nerve had been crushed half-way along its length at the time of explantation, monoclonal antibody 150 staining was absent from axons in the nerve proximal to the crush, but present in axons which had regenerated within the nerve distal to the crush. The results indicate that re-expression during axonal regeneration of the phosphorylated isoform of microtubule-associated protein 1B recognized by monoclonal antibody 150 is restricted to the newly formed lengths of regenerated axons. The correlation between its expression and axonal growth during development and regeneration suggests that it may play a role in axonal extension. Our observations also demonstrate the usefulness of these explant cultures for studying axonal regeneration

Expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in regenerating axons of the sciatic nerve

Monoclonal antibodies SMI-31 and 150 recognize phosphorylation epitopes on microtubule-associated protein 1B that have been shown to be developmentally down-regulated in the nervous system. We have used these antibodies to establish changes in the pattern of expression of their epitopes on microtubule-associated protein 1B in regenerating axons of the sciatic nerves in the adult mouse and rat. Immunohistochemical studies showed that, in the sciatic nerve, regenerating axons in both adult mice and rats were labelled with monoclonal antibody 150 in a proximodistal gradient which was highest at the growth cone. This is the first report of expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in regenerating axons. Immunoblotting showed that the expression of the isoform recognized by monoclonal antibody 150 is present in normal adult mouse sciatic nerve and in regenerating axons following crush or cut lesions, but was not detectable in the normal or regenerating adult rat peripheral nervous system. Regenerating axons were also labelled by monoclonal antibody SMI-31, but the labelling, unlike antibody 150 labelling, was uniform along the entire length of the axon and immunoblotting showed that it was due to recognition of neurofilament protein. We conclude that the phosphorylated isoforms of microtubule-associated protein 1B recognized by monoclonal antibody 150 that are developmentally down-regulated in the adult rat central and peripheral nervous systems and adult mouse cerebellum are maintained in the normal peripheral nervous system of the adult mouse. When peripheral axons regenerate in the adult mouse, the regenerating axons also contain these isoforms. Adult rat regenerating axons are stained by antibody 150 only in tissue sections, not in immunoblots. The maintenance of immature isoforms of microtubule-associated protein 1B in mouse peripheral axons may relate to a continual capacity for growth and remodelling. The immunohistochemical localization of the antibody 150 epitope in growth cone-like structures and sprouts in injured nerves shows that phosphorylation of microtubule-associated protein 1B is likely to be an integral part of the regenerative response. These results also show that the phosphorylation epitopes on microtubule-associated protein 1B recognized by monoclonal antibodies 150 and SMI-31 are different and that only expression of the former correlates with axonal regeneration

Evidence that glycogen synthase kinase-3 isoforms have distinct substrate preference in the brain

NoMammalian glycogen synthase kinase-3 (GSK3) is generated from two genes, GSK3alpha and GSK3beta, while a splice variant of GSK3beta (GSK3beta2), containing a 13 amino acid insert, is enriched in neurons. GSK3alpha and GSK3beta deletions generate distinct phenotypes. Here, we show that phosphorylation of CRMP2, CRMP4, beta-catenin, c-Myc, c-Jun and some residues on tau associated with Alzheimer's disease, is altered in cortical tissue lacking both isoforms of GSK3. This confirms that they are physiological targets for GSK3. However, deletion of each GSK3 isoform produces distinct substrate phosphorylation, indicating that each has a different spectrum of substrates (e.g. phosphorylation of Thr509, Thr514 and Ser518 of CRMP is not detectable in cortex lacking GSK3beta, yet normal in cortex lacking GSK3alpha). Furthermore, the neuron-enriched GSK3beta2 variant phosphorylates phospho-glycogen synthase 2 peptide, CRMP2 (Thr509/514), CRMP4 (Thr509), Inhibitor-2 (Thr72) and tau (Ser396), at a lower rate than GSK3beta1. In contrast phosphorylation of c-Myc and c-Jun is equivalent for each GSK3beta isoform, providing evidence that differential substrate phosphorylation is achieved through alterations in expression and splicing of the GSK3 gene. Finally, each GSK3beta splice variant is phosphorylated to a similar extent at the regulatory sites, Ser9 and Tyr216, and exhibit identical sensitivities to the ATP competitive inhibitor CT99021, suggesting upstream regulation and ATP binding properties of GSK3beta1 and GSK3beta2 are similar