Lester I. Binder

Proteolysis of tau by calpain

The calpain-induced proteolysis of tau associated with twice-cycled microtubules or from a total brain heat-stable fraction was studied. Twice-cycled microtubule tau was rapidly hydrolyzed by calpain. In contrast, tau purified from the total brain heat-stable fraction was very resistant to degradation by calpain. These results clearly demonstrate that there are at least 2 populations of tau in the brain based on calpain-sensitivity, a calpain-sensitive form that is associated with microtubules and a calpain-resistant form that may represent another population of tau in the brain.

Polymerization of microtubule-associated protein tau under near-physiological conditions.

Neurofibrillary tangles, which form in certain degenerating neurons in the brains of patients with Alzheimers disease, are amassed from filaments having a straight or paired helical morphology. Solubilization of these filaments reveals that they are composed of the microtubule-associated protein tau. It has not previously been shown, however, that tau will assemble to form filaments of similar morphology under conditions representative of the intracellular environment. We have succeeded in forming such filaments using tau purified from porcine or rat microtubules. The filaments are relatively straight with narrowing at irregular intervals, and are about 10 nm wide, a morphology similar to that of straight filaments seen in Alzheimers disease neurofibrillary tangles. At tau concentrations of 1-10 μM, in vitro assembly occurs at physiological pH, ionic strength, temperature, and reducing potential, and each one of these factors modulates the reaction. Assembly is judged to be only slowly reversible by the exponential rather than normal distribution of filament lengths, and by the limited disassembly observed under conditions which inhibit polymerization. Tau purified directly from whole brain tissue rather than from microtubules does not polymerize under conditions described in this report.

Phylogenetic conservation of brain microtubule-associated proteins MAP2 and tau.

The major rat brain microtubule-associated proteins, MAP2 and tau, exhibit various properties that implicate them in the mechanisms underlying the growth of axons and dendrites during neuronal development. To determine if these properties represent fundamental morphogenetic mechanisms, we have examined the phylogenetic conservation of these proteins in Xenopus laevis, quail and rat with respect to their molecular form, cytological distribution and developmental expression. In all three species, the high-molecular weight form of MAP2 migrates as a pair of polypeptides (MAP2a and MAP2b); this doublet as well as the low-molecular weight form of MAP2 (MAP2c) and the tau proteins are markedly similar in size in the different classes of vertebrates. Immunohistochemical staining of the Xenopus and quail cerebellum showed that MAP2 is highly concentrated in dendrites whereas the tau proteins are predominantly confined to axons, exactly as they are in rat. The developmental regulation of these proteins in Xenopus and rat is also conserved. Between the larva and the adult (i.e. during metamorphosis) MAP2c undergoes a marked decrease while MAP2a undergoes a large increase. Thus, in both classes of vertebrates the timing of changes in MAP2 expression coincides with the maturation of neuronal morphology. Taken together, these conserved properties of MAP2 and tau in three phylogenetically divergent classes of vertebrates suggest that these proteins serve fundamental functions during neuronal morphogenesis.

Differential localization of the high- and low-molecular weight variants of MAP2 in the developing retina

Microtubule-associated protein 2 (MAP2) occurs in developing mammalian neuronal tissue as both high (280 kDa)- and low (70 kDa)-molecular weight forms with temporally regulated expression. We have studied the developing avian retina with a monoclonal antibody that recognizes both the high- and low-molecular weight forms of MAP2 and a second monoclonal antibody that recognizes only high-molecular weight MAP2. The developmentally regulated, low-molecular weight protein, MAP2c, has a more widespread distribution in the embryonic avian retina than high-molecular weight MAP2. Our results suggest that MAP2c is the first form of MAP2 to appear in differentiated embryonic retinal neurons, and that the high-molecular weight isoforms of MAP2 appear only later when they may confer stability to neuronal processes.