Brain Microtubule Protein Research Articles

Glycation of rat sciatic nerve tubulin in experimental diabetes mellitus

Diabetic neuropathy is associated with some early defects of axonal transport in experimental animals. Axonal transport is dependent on intact microtubules, and unsubstituted lysine residues of tubulin are essential for microtubule polymerization. As lysine residues are the major target for the non-enzymatic attachment of glucose, the effect of diabetes on the extent of glycation of tubulin was investigated. There was a more than four-fold increase in the extent of glycation of tubulin in the sciatic nerve of rats with streptozotocin-induced diabetes of 2 weeks duration compared with control rats. In contrast, no such increase in glycation was observed in brain microtubule protein from diabetic rats at that stage of diabetes. Incubation of brain microtubule protein with glucose prior to in vitro polymerization showed that the early stages of glycation were not associated with inhibition of microtubule assembly. The observed glycation of peripheral nerve tubulin in early experimental diabetes may nevertheless contribute to axonal transport abnormalities through an as yet undetermined impairment of microtubule function.

Persistent alterations of calmodulin kinase II activity in chickens after an oral dose of tri- o-cresyl phosphate

Calmodulin kinase II has been found to be involved in the increased phosphorylation of brain microtubule and spinal cord neurofilament triplet proteins following treatment of animals with organophosphorus compounds that are capable of producing organophosphorus compound-induced delayed neurotoxicity (OPIDN). In this report, chickens were given a single oral neurotoxic dose of 750 mg/kg tri- o-cresyl phosphate (TOCP), and killed after 1 or 21 days of treatment. Crude calmodulin kinase II from brain cytosol as well as phosphocellulose-purified microtubules were prepared from control and treated animals. Phosphorylation reactions were started by adding protein into the phosphorylation buffer in the presence of Mg 2+, Ca 2+, calmodulin or trifluoperazine, and [γ- 32P]ATP. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to autoradiography. The extent of the calmodulin kinase II autophosphorylation as well as the Ca 2+/ calmodulin-dependent phosphorylation of the purified microtubules was investigated. The enzyme activities isolated from control and treated animals were compared. Autophosphorylation of calmodulin kinase II was found to be higher in both 1-day and 21-day TOCP-treated animals than in control animals. The activity of the kinase to phosphorylate exogenous substrates such as tubulin and microtubule-associated protein-2 (MAP-2) was also higher in the treated hens than in the controls. The increased activity of the kinase was noted at day 1 following treatment when no clinical signs were observed and persisted until day 21 when the animals were paralyzed completely. This finding supports the significance of altered calmodulin kinase II in the pathogenesis of OPIDN.

A calmodulin inhibitor with high specificity, compound 48/80, inhibits axonal transport in frog nerves without disruption of axonal microtubules

The calmodulin inhibitor compound 48/80 has previously been shown to arrest axonal transport in vitro in the regenerating frog sciatic nerve. The inhibition was limited to the outgrowth region of nerves, which had been allowed to regenerate in vivo for 6 days after a crush lesion, before they were incubated with or without drugs in vitro overnight. The effects of compound 48/80 on the regenerating nerve were further investigated. A concentration of compound 48/80 (50 micrograms ml-1), which effectively inhibits axonal transport, did not cause observable changes of the microtubules of regenerating axons in the outgrowth region as judged by electron microscopy. Furthermore, it was shown that also a lower concentration (25 micrograms ml-1) inhibited axonal transport. As a measure of possible metabolic effects, the level of ATP was assessed in the regenerating nerve after exposure to compound 48/80. Compound 48/80 at 25 micrograms ml-1 did not change the level of ATP in the nerve. The assembly of bovine brain microtubule proteins in a cell-free system was unaffected by 25 micrograms ml-1 of compound 48/80 and slightly inhibited by 50 micrograms ml-1. At higher concentrations (greater than 100 micrograms ml-1) assembly of microtubules appeared stimulated, and microtubule spirals as well as closely aligned microtubules could be seen. These effects appeared to be unrelated to the transport effects. The present results indicate that compound 48/80 arrests axonal transport via mechanisms other than destruction of axonal microtubules or interference with the energy metabolism. It is possible that these mechanisms involve inhibition of calmodulin-regulated events essential to the transport.

Microtubule-associated proteins-dependent colchicine stability of acetylated cold-labile brain microtubules from the Atlantic cod, Gadus morhua.

Assembly of brain microtubule proteins isolated from the Atlantic cod, Gadus morhua, was found to be much less sensitive to colchicine than assembly of bovine brain microtubules, which was completely inhibited by low colchicine concentrations (10 microM). The degree of disassembly by colchicine was also less for cod microtubules. The lack of colchicine effect was not caused by a lower affinity of colchicine to cod tubulin, as colchicine bound to cod tubulin with a dissociation constant, Kd, and a binding ratio close to that of bovine tubulin. Cod brain tubulin was highly acetylated and mainly detyrosinated, as opposed to bovine tubulin. When cod tubulin, purified by means of phosphocellulose chromatography, was assembled by addition of DMSO in the absence of microtubule-associated proteins (MAPs), the microtubules became sensitive to low concentrations of colchicine. They were, however, slightly more stable to disassembly, indicating that posttranslational modifications induce a somewhat increased stability to colchicine. The stability was mainly MAPs dependent, as it increased markedly in the presence of MAPs. The stability was not caused by an extremely large amount of cod MAPs, since there were slightly less MAPs in cod than in bovine microtubules. When "hybrid" microtubules were assembled from cod tubulin and bovine MAPs, these microtubules became less sensitive to colchicine. This was not a general effect of MAPs, since bovine MAPs did not induce a colchicine stability of microtubules assembled from bovine tubulin. We can therefore conclude that MAPs can induce colchicine stability of colchicine labile acetylated tubulin.

Differential in vitro action of S-12363, a new vinblastine derivative, and of its epimer on microtubule proteins

The action of two epimers of a new vinblastine derivative that differ in their in vivo antitumor activity and their cytotoxicity was studied in vitro in brain microtubule proteins. These two compounds, called S-12363 and S-12362, could not be distinguished from one another or from other active vinca alkaloids by their ability to prevent microtubule assembly. However, they differed strongly both from one another and from vincristine and vinblastine in their ability to induce the formation of tubulin paracrystals and in the stability of the paracrystals following temperature shifts from 0 degree to 37 degrees C and vice versa. The most potent drug, S-12363, induced considerable tubulin aggregation, which was even more pronounced than that observed in the presence of vincristine. Previous results have shown that S-12363, in contrast to vincristine, induces no neurotoxic effects. This observation is in disagreement with a direct relationship between tubulin aggregation and neurotoxicity.

The dynamic instability of microtubules is not modulated by α‐tubulin tyrosinylation

The tyrosinylation of chick brain alpha-tubulin and the effects of the tyrosinylation status on the assembly and dynamic instability of chick brain MAP2:tubulin microtubule protein have been examined. Each of the eight major alpha-isotypes can be tyrosinylated in vitro, irrespective of whether a C-terminal tyrosine is genetically encoded. The extent of tyrosinylation is however limited to congruent to 0.3 mol.mol-1. The tyrosinylation status (0 vs. 0.3 mol.mol-1) has no effect on either the assembly kinetics of chick brain microtubule protein or on the rate of length redistribution following assembly and shearing. It is therefore unlikely that the tyrosinylation status directly affects the intrinsic stability of assembled microtubules since the rate of length redistribution is both a sensitive assay and a function of the kinetic parameters governing dynamic instability.

Proteolytic cleavage of microtubule-associated proteins by retroviral proteinases

Aspartic proteinases from human immunodeficiency virus type 1 (HIV-1) and avian myeloblastosis virus (AMV) were found to interfere with microtubule assembly. Preincubation of the proteinases with purified brain microtubule proteins (tubulin and microtubule-associated proteins) at low ionic strength (pH 6.8), completely inhibited microtubule assembly. Analysis of microtubule proteins after incubation with proteinase showed no effect on tubulin but extensive cleavage of the microtubule-associated proteins 1 and 2 was observed. The digestion by the two proteinases differed. In the presence of HIV-1 proteinase, a fragment with an Mr of approximately 300, appeared, as well as at least three other new fragments, with Mr values of 188,000, 124,000 and 73,000. In the presence of AMV proteinase, the microtubule-associated proteins were extensively digested to many small fragments. The extending microtubule-associated proteins normally seen by electron microscopy on the microtubule surface disappeared after treatment with AMV proteinase. Our results show that retroviral proteinases are not restricted to cleavage of viral polyproteins in vitro. It is suggested that proteolysis of microtubular proteins by viral proteinases is an important step in viral pathogenicity and that it may be part of a mechanism causing degenerative effects in infected cells.

A Nerve Growth Factor‐Dependent Protein Kinase That Phosphorylates Microtubule‐Associated Proteins In Vitro: Possible Involvement of Its Activity in the Outgrowth of Neurites from PC12 Cells

We have established a subline of PC12 cells (PC12D) that extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP (cAMP) in the same way as primed PC12 cells (NGF-pretreated cells). When phosphorylation of brain microtubule proteins by extracts of these cells was monitored, two distinct kinase activities were found to be increased [from three-to eightfold in terms of phosphorylation of microtubule-associated protein (MAP) 2] by a brief exposure of cells to NGF or to dibutyryl cAMP (dbcAMP). The effect of the combined stimulation with both NGF and dbcAMP was additive in terms of the phosphorylation of MAP2. The apparent molecular mass of the kinase activated by dbcAMP was 40 kDa, and this kinase appears to be cAMP-dependent protein kinase. The molecular mass of the kinase activated by NGF was 50 kDa. The latter was activated to a measurable extent after 5 min of exposure of cells to NGF: it required Mg2+ for activity but not Mn2+ or Ca2+. This kinase appears to be distinct from previously reported kinases in PC12 cells, and it has been designated as NGF-dependent MAP kinase, although its physiological substrates are not known at present. An inhibitor of protein kinases, K-252a, selectively inhibited the outgrowth of neurites from PC12D cells in response to NGF but not to dbcAMP. When this inhibitor was added to the incubation medium of cells exposed simultaneously to NGF or dbcAMP, the increase in activity of the NGF-dependent MAP kinase was selectively abolished.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of Microtubule Proteins in Rat Brain at Different Developmental Stages: Comparison with That Found in Neuronal Cultures

The phosphorylation of rat brain microtubule protein on intracranial injection of labeled phosphate has been analyzed. The major microtubule protein components phosphorylated in vivo in rat brain are the high-molecular-weight microtubule-associated proteins (MAPs) MAP-1A, MAP-1B, and MAP-2. A slight phospholabeling of beta-tubulin, which corresponds to the phosphorylation of a minor neuronal beta-tubulin isotype, is also observed. Whereas MAP-1B, MAP-2, and beta-tubulin are phosphorylated in the brain of 5-day-old rat pups, when most neurons of the CNS are extending processes, MAP-1A phosphorylation is observed only after neuronal maturation takes place. The phosphorylation of MAP-1A, MAP-1B, and beta-tubulin may be due mainly to casein kinase II or a related enzyme, whereas MAP-2 appears to be modified by other enzymes such as the cyclic AMP-dependent protein kinase (protein kinase A) and the calcium/phospholipid-dependent protein kinase (protein kinase C). Microtubule protein phosphorylation has also been studied in neuronal cultures. In differentiated neuroblastoma cells, only MAP-1B and beta-tubulin are phosphorylated in a manner coupled to neurite outgrowth. In primary cultures of fetal rat brain neurons, the pattern of microtubule protein phosphorylation resembles that found in vivo in rat pup brain. As phosphorylated MAP-1A and MAP-1B are present mainly on assembled microtubules, whereas the phosphorylation of MAP-2 decreases its interaction with microtubules, a role can be suggested for the phosphorylation of these proteins in the regulation of microtubule assembly and disassembly during neuronal development.

A novel heat-stable 205 kDa microtubule-associated protein is involved in the neural development of the rat brain

A novel microtubule associated protein (MAP) was identified with a monoclonal antibody (mAb) among rat brain microtubule (MT) proteins. This MAP, which is found in a heat-stable MAP fraction, is co-polymerized with tubulin from rat brain homogenates both in cycled and in taxol-driven assembly purifications. When the heat-stable fraction of MT proteins and purified tubulin are assembled in vitro without taxol, 205 kDa MAP is identified in the MT pellet. Immunofluorescent studies using the mAb against this MAP on sections of a rat spinal cord, hippocampus, retina and cerebellar cortex, showed that 205 kDa MAP was localized in not only neural cell bodies, dendrites, and axons, but also in glial cells. This 205 kDa MAP is clearly distinct from previously reported MAPs as to molecular mass, heat stability, immunoreactivity, distribution among tissues or within a neuron, and the developmental aspect of the postnatal cerebellar cortex. As for development, the 205 kDa MAP has already been seen to appear at postnatal day 3. Throughout the postnatal development of the cerebellar cortex, 205 kDa MAP is expressed in developing Purkinje cells, with the staining reaction being particularly intense in the more superficially positioned parallel fiber profiles. This points out the possible important functions of 205 kDa MAP in the morphogenesis of parallel fiber axons.

Specific alterations in the biological activities of C-20'-modified vinblastine congeners

Both the anti-tumor and toxic activities of the vinca alkaloid dimers, vinblastine (VBL) and vincristine (VCR), may reside at the level of their known cellular target, the microtubule system. The contributions made by each of the various actions of these alkaloids on this system are unknown. We have used new, complete synthetic methodologies to create a series of eight C-20' alkyl congeners of VBL and have examined these compounds for their abilities to (1) inhibit microtubule assembly, (2) disassemble preformed microtubules, and (3) induce spiral aggregate formation, using purified brain microtubule protein. By combining turbidimetric and electron microscopic techniques, we discovered that each of the various effects of VBL on the microtubule system in vitro was amenable to alteration by specific modification at this single molecular site. In addition, we report two new aberrations of VBL action—the induction of spirals by a concentration of congener below 1 μM and the formation of “opened” microtubules polymerized in the presence of congener. The relationship between anti-microtubule action in vitro and the cellular activities of growth inhibition and mitotic arrest by the congeners was examined in leukemic and colon cancer cell lines. In general, we found that both cellular perturbations were correlated to the ability of the congeners to inhibit microtubule polymerization rather than to the actions of spiral formation or microtubule disassembly. These results are a breakthrough in the structure/function relationship of the vinca alkaloid dimers and should provide the means to determine the role of specific anti-microtubule activities to the complex biological actions of these natural product drugs.

Calmodulin modulation of adverse effects of Cd 2+ on microtubules and tubulin polymerization in vitro

The contribution of calmodulin to the effects of Cd 2+ on microtubules has been investigated in the detergent-extracted cytoskeletons of cultured cells and on tubulin polymerization in vitro. When incubated with micromolar concentrations of Cd 2+ or Ca 2+ alone, or a mixture of both ions, microtubules in the extracted cytoskeletons disassembled. This disassembly was prevented by compound 48/80, a potent calmodulin inhibitor. In bovine brain microtubule protein containing microtubule-associated proteins, calmodulin also enhanced the Cd 2+-mediated inhibition of tubulin polymerization; further, this enhancing effect of calmodulin was reversed by compound 48/80. These results demonstrate that Cd 2+ can affect microtubules by binding to and activating calmodulin.

A Novel Tubulin-Dependent Protein Kinase Forming a Paired Helical Filament Epitope on Tau

From rat brain microtubule proteins, we purified a protein kinase that phosphorylated tau, one of microtubule-associated proteins. The electrophoretic mobility of the phosphorylated tau on SDS-polyacrylamide gel decreased. The enzyme was not activated by cyclic nucleotides, calmodulin, or phospholipids, and was inhibited by the calcium ions. The kinase bound to tau. The phosphorylation of tau was stimulated by tubulin under the condition of microtubule formation. From these results we propose an idea that the phosphorylation could occur concomitantly with microtubule formation in the brain. Human tau phosphorylated by the kinase carried an epitope of the paired helical filaments that accumulate in the brain in Alzheimer's disease.

A casein kinase II-related activity is involved in phosphorylation of microtubule-associated protein MAP-1B during neuroblastoma cell differentiation.

A neuroblastoma protein related to the brain microtubule-associated protein, MAP-1B, as determined by immunoprecipitation and coassembly with brain microtubules, becomes phosphorylated when N2A mouse neuroblastoma cells are induced to generate microtubule-containing neurites. To characterize the protein kinases that may be involved in this in vivo phosphorylation of MAP-1B, we have studied its in vitro phosphorylation. In brain microtubule protein, MAP-1B appears to be phosphorylated in vitro by an endogenous casein kinase II-like activity which also phosphorylates the related protein MAP-1A but scarcely phosphorylates MAP-2. A similar kinase activity has been detected in cell-free extracts of differentiating N2A cells. Using brain MAP preparations devoid of endogenous kinase activities and different purified protein kinases, we have found that MAP-1B is barely phosphorylated by cAMP-dependent protein kinase, Ca/calmodulin-dependent protein kinase, or Ca/phospholipid-dependent protein kinase whereas MAP-1B is one of the preferred substrates, together with MAP-1A, for casein kinase II. Brain MAP-1B phosphorylated in vitro by casein kinase II efficiently coassembles with microtubule proteins in the same way as in vivo phosphorylated MAP-1B from neuroblastoma cells. Furthermore, the phosphopeptide patterns of brain MAP-1B phosphorylated in vitro by either purified casein kinase II or an extract obtained from differentiating neuroblastoma cells are identical to each other and similar to that of in vivo phosphorylated neuroblastoma MAP-1B. Thus, we suggest that the observed phosphorylation of a protein identified as MAP-1B during neurite outgrowth is mainly due to the activation of a casein kinase II-related activity in differentiating neuroblastoma cells. This kinase activity, previously implicated in beta-tubulin phosphorylation (Serrano, L., J. Díaz-Nido, F. Wandosell, and J. Avila, 1987. J. Cell Biol. 105: 1731-1739), may consequently have an important role in posttranslational modifications of microtubule proteins required for neuronal differentiation.

Calcium-independent, pH-regulated effects of S-100 proteins on assembly-disassembly of brain microtubule protein in vitro.

At alkaline pH, Ca2+ is no longer required for S-100 proteins to inhibit the assembly and to promote the disassembly of brain microtubules in vitro, though the presence of Ca2+ significantly favors the S-100 effects. These effects are inversely related to the microtubule protein concentration and directly related to the S-100 concentration and the pH. Ca2+-independent, pH-regulated inhibition of assembly of phosphocellulose-purified tubulin by S-100 is also described. The microtubule disassembling effect of S-100 is additive to that of alkali (used to raise the pH), and S-100 further disassembles microtubules after alkalinization. Thus the larger inhibitory effect of S-100 on microtubule assembly at alkaline versus acid pH depends on both a decrease in the assembly rate and an increase in the disassembly rate. Together with previous data on this topic, the present findings indicate that S-100 proteins act on microtubule protein in vitro primarily by binding to tubulin, this event being Ca2+-regulated at a given pH, and pH-regulated at a given free Ca2+ concentration.

Differential phosphorylation of microtubule proteins by ATP and GTP.

Purified brain microtubule protein is phosphorylated by endogenous protein kinase activities in the presence of [gamma-32P] ATP or [gamma-32P] GTP. Here we show that certain microtubule-associated proteins are phosphorylated differently by GTP or ATP as direct phosphoryl donors, suggesting the presence of distinct kinase activities, with different specificities, associated with microtubule protein.

Developmental regulation of two microtubule-associated proteins (MAP2 and MAP5) in the embryonic avian retina.

Previous studies with the mammalian brain have shown that the expression of a number of neuronal microtubule-associated proteins (MAPs) is developmentally regulated. For example, the low-molecular-weight form of MAP2 (MAP2c) is abundant in neonatal rat brains and is less abundant in adults. Similarly, MAP5 levels decrease during postnatal development. Using monoclonal antibodies, we have followed the time of first appearance, cellular distribution, and molecular form of MAP2 and MAP5 during the morphogenesis of the quail retina. MAP2 first appears in ganglion cell bodies and in the axons of the optic fibre layer (OFL) at embryonic day 4 (E4). Anti-MAP2 staining remains restricted to these sites until E10, when staining appears in the inner plexiform layer (IPL). At E14, one day before hatching, anti-MAP2 staining is found in three broad laminae in the IPL, as well as in photosensitive cells. MAP5 is present in ganglion cell axons from the onset of neurite elongation at E3 and is limited to the OFL until E10. The intensity of anti-MAP5 staining in the OFL and optic nerve decreases after E7, which corresponds with a decrease in the number of actively growing ganglion cell axons. By E14, anti-MAP5 stains five layers in the IPL that correspond with layers of amacrine cell process arborizations. Western blots of E10 brain microtubule proteins show that MAP2 is represented by both a 260 x 10(3) Mr protein and a 60-65 x 10(3) Mr protein; the latter is much more abundant. Anti-MAP5 recognizes a 320 x 10(3) Mr brain microtubule protein in both the quail and the rat. We conclude that the cellular distribution, developmental regulation and molecular forms of MAP2 and MAP5 are similar in the rat and quail, suggesting that these molecules have conserved and hence fundamental roles in the growth and differentiation of neuronal processes.

Tubulin phosphorylation by casein kinase II is similar to that found in vivo.

Purified brain tubulin subjected to an exhaustive phosphatase treatment can be rephosphorylated by casein kinase II. This phosphorylation takes place mainly on a serine residue, which has been located at the carboxy-terminal domain of the beta-subunit. Interestingly, tubulin phosphorylated by casein kinase II retains its ability to polymerize in accordance with descriptions by other authors of in vivo phosphorylated tubulin. Moreover, the V8 phosphopeptide patterns of both tubulin phosphorylated in vitro by casein kinase II and tubulin phosphorylated in vivo in N2A cells are quite similar, and different from that of tubulin phosphorylated in vitro by Ca/calmodulin-dependent kinase II. On the other hand, we have found an endogenous casein kinase II-like activity in purified brain microtubule protein that uses GTP and ATP as phosphate donors, is inhibited by heparin, and phosphorylates phosphatase-treated tubulin. Thus it appears that a casein kinase II-like activity should be considered a candidate for the observed phosphorylation of beta-tubulin in vivo in brain or neuroblastoma cells.

Characteristics of the effect of S-100 proteins on the assembly-disassembly of brain microtubule proteins at alkaline pH in vitro

The ability of S-100 proteins to inhibit the assembly of brain microtubule proteins (MTPs) in the presence of μM levels of Ca 2+ increases as a function of pH. This seems to be due to an increasingly larger inhibitory effect of S-100 on the nucleation and, probably, on the elongation of microtubules (MTs) as the pH raises. In the presence of μM Ca 2+ levels, the ability of S-100 to disassemble MTs also increases linearly with the pH, suggesting that the larger inhibitory effect of S-100 on MTP assembly at alkaline than at acidic pH may depend on both a decrease in the assembly rate and an increase in the disassembly rate. Also, S-100 inhibits the assembly of phosphocellulose-purified tubulin to a larger and larger extent as the pH raises. S-100 brings about its effect on MT assembly-dis assembly probably by sequestering soluble tubulin, though additional mechanisms cannot be excluded. The present data are briefly discussed in relation to the role attributed to changes in intracellular pH in the regulation of the state of assembly of cytoplasmic MTs.

Quantitative analysis of the interaction between S-100 proteins and brain tubulin

S-100 was shown to regulate the in vitro assembly of brain microtubule proteins (MTPs) in a Ca 2+-mediated way by acting on both the nucleation and the elongation of microtubules (MTs). Here data will be shown suggesting that S-100 binds to tubulin. The binding is time-, temperature-, Ca 2+-, and pH-dependent, and saturable with respect to S-100. At pH 6.75, the saturation curve is biphasic, displaying a high affinity component (dissociation constant, K d1,∼0.1 μM) and a low affinity component (K d2 ∼ 3.8 μM). At pH 6.75, as the free Ca 2+ concentration raises from 0 to 100 μM, the overall binding capacity increases from 0.065 to 0.66 mol S-100/mol tubulin dimer. This finding, together with the observation that the S-100 effect on MTP assembly is Ca 2+-dependent at that pH, suggests that the S-100-induced inhibition of MTP assembly depends on S-100 binding to the low affinity sites on the tubulin molecule. The S-100 binding to tubulin is pH-dependent: as the pH raises from 6.75 to 8.3, both binding components are affected, the major changes consisting of an increase in the binding capacity and a decrease in the overall affinity. Moreover, as the pH raises, Ca 2+ is no longer required for S-100 to bind to tubulin. S-100 also interacts with a component of whole MTPs (probably tubulin, on the basis of the above results). No S-100 binding to microtubule-associated proteins (MAPs) could be evidenced by the techniques employed in this study. On the contrary, some competition between S-100 and MAPs for binding sites or tubulin seems to occur.