Distribution and Subcellular Localization of High-Molecular-Weight Microtubule-Associated Protein-2 Expressing Exon 8 in Brain and Spinal Cord

Abstract: The expression of high-molecular-weight (HMW) microtubule-associated protein-2 (MAP-2) expressing exon 8 (MAP-2+8) was examined by immunoblotting during rat brain development and in sections of human CNS. In rat brain, HMW MAP-2+8 expression was detected at embryonic day 21 and increased during postnatal development. In adult rats, HMW MAP-2+8 comigrated with MAP-2a. In human adult brain, HMW MAP-2+8 was expressed in select neuronal populations, including pyramidal neurons of layers III and V of the neocortex and parahippocampal cortex, pyramidal neurons in the endplate, CA2 and subiculum of the hippocampus, and the medium-sized neurons of the basal ganglia. In the cerebellum, a subpopulation of Golgi neurons in the internal granular cell layer and most Purkinje cells were also stained. In the spinal cord staining was observed in large neurons of the anterior horn. Staining was present in cell bodies and dendrites but not in axons. At the ultra-structural level, HMW MAP-2+8 immunoreactivity was observed on mitochondrial membranes and in postsynaptic densities (PSDs) of some asymmetric synapses in the midfrontal cortex and spinal cord. Immunoblots of proteins isolated from enriched mitochondrial and PSD fractions from adult human frontal lobe and rat brains confirmed the presence of HMW MAP-2+8. The presence of HMW MAP-2+8 in dendrites and in close proximity to PSDs supports a role in structural and functional attributes of select excitatory CNS synapses.     

Human Microtubule-Associated Protein-2c Localizes to Dendrites and Axons in Fetal Spinal Motor Neurons

Abstract: Microtubule-associated protein-2 (MAP-2) functions to maintain neuronal morphology by promoting the assembly of microtubules. MAP-2c is an alternately spliced form of MAP-2, containing the first 151 amino acids of high-molecular-weight (HMW) MAP-2 joined to the last 321 amino acids, eliminating 1,352 amino acids specific to HMW MAP-2. A polyclonal antibody generated to the splice site of human MAP-2c was used to determine its cellular localization. The MAP-2c antiserum was depleted of any HMW MAP-2 reactivity by absorption with HMW MAP-2 fusion protein. Western blot analysis of human fetal spinal cord homogenates demonstrated that the antibody is specific for human MAP-2c. MAP-2c immunoreactivity was found in the perinuclear cytoplasm and processes of anterior motor neurons and large processes of the posterior column in sections from 22–24-week human fetal spinal cord. Double-label confocal microscopy was performed using the MAP-2c polyclonal antibody and either a HMW MAP-2 or a neurofilament protein (highly phosphorylated 160- and 200-kDa protein) monoclonal antibody to identify these processes as dendrites or axons, respectively. HMW MAP-2 and MAP-2c colocalized in cell bodies and dendrites of anterior motor neurons, demonstrating for the first time the presence of native MAP-2c within dendrites. In addition, immunoelectron microscopy showed MAP-2c associated with microtubules in dendrites of motor neurons. MAP-2c and the neurofilament proteins were found in axons of the dorsal and ventral roots. The presence of MAP-2c within axons and dendrites suggests that MAP-2c contributes to neuronal plasticity during human fetal development.     

A novel microtubule-associated protein-2 expressed in oligodendrocytes in multiple sclerosis lesions

Elucidation of the mechanisms involved in the regeneration of oligodendrocytes and remyelination is a central issue in multiple sclerosis (MS) research. We recently identified a novel alternatively spliced, developmentally regulated oligodendrocyte-specific protein designated microtubule-associated protein-2+13 [microtubule-associated protein-2 expressing exon 13 (MAP-2+13)]. MAP-2+13 is expressed in human fetal oligodendrocytes during process extension and myelination but is minimally expressed in normal mature CNS. To test the hypothesis that MAP-2+13 is reexpressed in regenerating oligodendrocytes in MS lesions, we examined the brains of MS patients for the expression of this protein. By immunocytochemistry using a series of monoclonal antibodies specific for MAP-2+13, we determined that MAP-2+13 expression was up-regulated in all 31 lesions from 10 different MS brains. MAP-2+13 was expressed in regenerating oligodendrocytes associated with demyelinated lesions, with the highest counts found in regions of extensive remyelination. By electron microscopy, MAP-2+13 was localized to oligodendrocytes engaged in remyelination, evident by their process extension and association with thinly myelinated (remyelinated) and demyelinated axons. These results suggest a hitherto unsuspected role for this microtubule-associated protein in oligodendrocyte function during development and myelin repair.     

Proteolysis of Microtubule-Associated Protein 2 and Tubulin by Cathepsin D

The in vitro degradation of microtubule-associated protein 2 (MAP-2) and tubulin by the lysosomal aspartyl endopeptidase cathepsin D was studied. MAP-2 was very sensitive to cathepsin D-induced hydrolysis in a relatively broad, acidic pH range (3.0-5.0). However, at a pH value of 5.5, cathepsin D-mediated hydrolysis of MAP-2 was significantly reduced and at pH 6.0 only a small amount of MAP-2 was degraded at 60 min. Interestingly, the two electrophoretic forms of MAP-2 showed different sensitivities to cathepsin D-induced degradation, with MAP-2b being significantly more resistant to hydrolysis than MAP-2a. To our knowledge, this is the first clear demonstration that MAP-2 is a substrate in vitro for cathepsin D. In contrast to MAP-2, tubulin was relatively resistant to cathepsin D-induced hydrolysis. At pH 3.5 and an enzyme-to-substrate ratio of 1: 20, only 35% of the tubulin was degraded by cathepsin D at 60 min. The cathepsin D-mediated hydrolysis of tubulin was optimal only at pH 4.5. These results demonstrate that MAP-2 and tubulin are unequally susceptible to degradation by cathepsin D. These data also imply a potential for rapid degradation of MAP-2 in vivo by cathepsin D either in lysosomes or perhaps autophagic vacuoles of the neuron.     

Localization of Specific Epitopes on Human Microtubule-Associated Protein 2

Abstract: Microtubule-associated protein 2 (MAP-2) is an abundant neuronal cytoskeletal protein that binds to tubulin and stabilizes microtubules. Using fusion protein constructs we have defined the epitopes of 10 monoclonal antibodies (mAbs) to discrete regions of human MAP-2. Proteins were expressed in pATH vectors. After electrophoresis, immunoblotting was performed. By western blot analysis five of the mAbs (AP-14, AP-20, AP-21, AP-23, and AP-25) share epitopes with only the high molecular weight isoforms (MAP-2a, MAP-2b); two of the mAbs (AP-18 and tau 46) recognize MAP-2a, MAP-2b, and MAP-2c. Although AP-18 immunoreactivity was detected within heat-stable protein homogenates isolated from a human neuroblastoma cell line MSN, fusion protein constructs encompassing human MAP-2 were negative, suggesting that the AP-18 epitope is phosphorylated. Furthermore, AP-18 immunoreactivity was lost after alkaline phosphatase treatment of heat-stable protein preparations from MSN cells. Four of the mAbs (322, 636, 635, and 39) recognize epitopes located within amino acids 169–219 of human MAP-2. AP-21 maps to a region between amino acids 553 and 645. AP-23 maps between amino acids 645 and 993, whereas AP-20, AP-14, and AP-25 map between amino acids 995 and 1332. Expression of the region of MAP-2 between amino acids 1787 and 1824 was positive to tau 46.     

Activation of a Microtubule-Associated Protein-2 Kinase by Insulin-Like Growth Factor-I in Bovine Chromaffin Cells

Treatment of bovine chromaffin cells with insulin-like growth factor-I (IGF-I) caused the activation of a protein kinase that phosphorylates microtubule-associated protein-2 (MAP-2) in vitro. Activation of MAP-2 kinase by IGF-I varied with the time of treatment (maximal at 10-15 min) and the concentration of IGF-I (maximal at 10 nM). The IGF-I-activated MAP-2 kinase was localized to the soluble fraction of chromaffin cell extracts and required Mg2+ for activity. The IGF-I-activated kinase also phosphorylated myelin basic protein, but had little or no activity toward histones or ribosomal S6 protein. To examine the role of protein tyrosine phosphorylation in the activation of the MAP-2 kinase, we isolated phosphotyrosine (PTyr)-containing proteins from chromaffin cells by immunoaffinity adsorption on anti-PTyr-Sepharose beads. Anti-PTyr-Sepharose eluates from IGF-I-treated cells showed increased MAP-2 kinase activity; thus, the MAP-2 kinase (or a closely associated protein) appears to be a PTyr-containing protein. Treatment of anti-PTyr-Sepharose eluates or crude chromaffin cell extracts with alkaline phosphatase significantly decreased kinase activity toward myelin basic protein, indicating that phosphorylation of the IGF-I-activated kinase is required for its activity.     

Microtubule-Associated Protein 2 as an Early Indicator of Ischemia-Induced Neurodegeneration in the Gerbil Forebrain

Abstract: Microtubule-associated protein 2 (MAP-2) was studied in the gerbil hippocampus and striatum after transient ischemia. Western immunoblot analysis shows that there is a significant decrease of MAP-2 in the dorsolateral sector of the striatum and a slight decrease of MAP-2 in the CA1 region of the hippocampus 6–12 h after ischemia in the gerbil forebrain. The immunohistochemical staining pattern of MAP-2 in these two regions also shows a loss of immunostaining of MAP-2. In particular, a beaded MAP-2 immunostaining pattern at the apical dendritic region of the CA1 neurons of the hippocampus was found within 12 h after ischemia compared with the smooth dendritic immunostaining of MAP-2 in normal CA1 neurons. In vitro assays of MAP-2 degradation suggest that dendritic loss of immunoreactivity after ischemia seen on western blots may be due to calpain I degradation of MAP-2. Loss of MAP-2 in both the striatum and hippocampus was found to occur earlier than spectrin degradation by western blot analysis. These results suggest that loss of MAP-2 may participate in the initial phase of neuronal dysfunction and that dendritic breakdown may be a first sign of neurodegeneration.     

Degradation of Microtubule-Associated Protein 2 and Brain Spectrin by Calpain: A Comparative Study

The in vitro degradation of microtubule-associated protein 2 (MAP-2) and spectrin by the calcium-dependent neutral protease calpain was studied. Five major results are reported. First, MAP-2 isolated from twice-cycled microtubules (2 X MT MAP-2) was extremely sensitive to calpain-induced hydrolysis. Even at an enzyme-to-substrate ratio (wt/wt) of 1:200, 2 X MT MAP-2 was significantly degraded by calpain. Second, MAP-2 purified from the total brain heat-stable fraction (total MAP-2) was significantly more resistant to calpain-induced hydrolysis compared with 2 X MT MAP-2. Third, MAP-2a and MAP-2b were proteolyzed similarly by calpain, although some relative resistance of MAP-2b was observed. Fourth, the presence of calmodulin significantly increased the extent of calpain-induced hydrolysis of the alpha-subunit of spectrin. Fifth, the two neuronal isoforms of brain spectrin (240/235 and 240/235E, referred to as alpha/beta N and alpha/beta E, respectively) showed different sensitivities to calpain. alpha N-spectrin was significantly more sensitive to calpain-induced degradation compared to alpha E-spectrin. Among other things, these results suggest a role for the calpain-induced degradation of MAP-2, as well as spectrin, in such physiological processes as alterations in synaptic efficacy, dendritic remodeling, and in pathological processes associated with neurodegeneration.     

Ammonium Injection Induces an N-Methyl-d-Aspartate Receptor-Mediated Proteolysis of the Microtubule-Associated Protein MAP-2

Abstract: We have shown previously that chronic hyperammonemia increases, in brain, the polymerization of microtubules that is regulated mainly by the level and state of phosphorylation of microtubule-associated protein 2 (MAP-2). Activation of the N -methyl- d -aspartate (NMDA) receptor dephosphorylates MAP-2. Because we have found that acute ammonia toxicity is mediated by the NMDA receptor, we have tested the effect of high ammonia levels on MAP-2 in brain. Microtubules isolated from rats injected intraperitoneally with 6 mmol/kg ammonium acetate showed a marked decrease of MAP-2. Also, the amount of MAP-2 in brain homogenates, determined by immunoblotting. was markedly reduced, presumably by proteolysis. The content of MAP-2 was decreased by ∼75% 1-2 h after ammonium injection and returned to normal values after 4 h. Proteolysis of MAP-2 was prevented completely by injection of 2 mg/kg MK-801, a specific antagonist of the NMDA receptor, suggesting that proteolysis is mediated by activation of this receptor. l -Carnitine, which protects rats against ammonia toxicity, also prevented MAP-2 degradation. Because activation of the NMDA receptor increases [Ca²⁺]_i, we determined whether rat brain contains a Ca²⁺-dependent protease that selectively degrades MAP-2. We show that there is a cytosolic Ca²⁺-dependent protease that degrades MAP-2, but no other brain proteins. The protease has been identified tentatively as calpain I, for it is inhibited by a specific inhibitor of this protease. Our results suggest that ammonium injection activates the NMDA receptor, leading to an increase in [Ca²⁺]_i, which activates calpain I. This, in turn, selectively degrades MAP-2. Possible implications in chronic hyperammonemic states and in the mechanism of ammonia toxicity are discussed.     

Cleavage of Bovine Brain Microtubule-Associated Protein-2 by Human Immunodeficiency Virus Proteinase

The high-molecular-weight dendritic cytoskeletal protein known as microtubule-associated protein (MAP)-2 displays the capacity to stimulate tubulin polymerization and to associate with microtubules. Serine proteases cleave MAP-2 into a C-terminal M(r) 28,000-35,000 microtubule-binding fragment and a larger N-terminal M(r) 240,000 projection-arm region. We now show that human immunodeficiency virus (HIV) proteinase also progressively degrades purified MAP-2 in vitro. This proteolysis reaction is characterized by transient accumulation of at least six intermediates, and most abundant of these is an M(r) 72,000 species that retains the ability to associate with taxol-stabilized microtubules. Treatment of this M(r) 72,000 species with thrombin releases the same M(r) 28,000 component as that derived from thrombin action on intact high-molecular-weight MAP-2, indicating that the viral aspartoproteinase action preferentially occurs further toward the N-terminus. The association of the M(r) 72,000 component with microtubules can be disrupted by the presence of a 21-amino acid peptide analogue of the second repeated sequence in the MAP-2 microtubule-binding region. We also studied HIV proteinase action on MAP-2 in the presence of tubulin and other MAPs that recycle with tubulin, and contrary to other published studies we found no effect of such treatment on microtubule self-assembly behavior. Cleavage of isolated MAP-2 by the HIV enzyme at high salt concentrations, followed by desalting and addition of tubulin, also resulted in microtubule assembly, albeit with slightly reduced efficiency.     

Polyamine Regulation of the Microtubule-Associated Protein Kinase

Microtubule protein prepared by cycles of assembly-disassembly contains a cyclic AMP-dependent protein kinase that phosphorylates the high-molecular-weight microtubule-associated protein MAP-2. The polyamine spermine at 2mM affected the phosphorylation of MAP-2 in a manner that depended on the cyclic AMP concentration. At cyclic AMP concentrations below 10(-6) M, spermine increased the rate of phosphorylation, while at cyclic AMP concentrations above 10(-6) M, spermine decreased the rate of phosphorylation. Spermine also decreased the final extent of cyclic AMP-dependent phosphorylation but did not affect the protein substrate specificity of the microtubule-associated protein kinase. MAP-2 was the principal substrate both in the presence and in the absence of spermine. Because of these results, we propose that microtubule protein phosphorylation may be regulated in vivo by spermine as well as by cyclic AMP levels.     

Induction of cytotoxic T lymphocytes specific to malignant glioma using T2 cells pulsed with HLA-A2-restricted interleukin-13 receptor alpha 2 peptide in vitro

Interleukin-13 receptor α2 （IL-13Rα2） is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. The present study is a report of a novel approach of targeting malignant glioma with IL-1 3Rα2-specific cytotoxic T lymphocyte （CTL） induced from the peripheral blood mononuclear cells of healthy donors by multiple stimulations with human leukocyte antigen （HLA）-A2-restricted IL-1 3Rα2345-353 peptide-pulsed T2 cells. The induced CTL showed specific lysis against T2 cells pulsed with the peptide and HLA-A2^＋ glioma cells expressing IL- 1 3Rα2345-353, while HLA-A2 glioma cell lines that express IL-13Rα2345-353 could not be recognized by CTL. The peptide-specific activity was inhibited by anti-HLA class I monoclonal antibody. These results suggest that the induced CTL specific for IL-1 3Rα2345-353 peptide could be a potential target of specific immunotherapy for HLA-A2 patients with malignant glioma.     

MAP2a, an Alternatively Spliced Variant of Microtubule-Associated Protein 2

Abstract: MAP2, a dendritically localized microtubule-associated protein (MAP), consists of a pair of high molecular mass (280 kDa) polypeptides, MAP2a and MAP2b, and several low molecular mass (70 kDa) proteins called MAP2c. Although MAP2b and MAP2c have been shown to arise via alternative splicing, it was not clear whether MAP2a is also created by alternative splicing or by posttranslational modification. Using epitope peptide mapping, we have demonstrated that an element specific to MAP2a is situated at its N-terminal end. A cDNA clone from an adult rat brain library was found to contain an additional 246 nucleotides situated at the 5' end of the 9-kb MAP2 mRNA. Antibodies generated against the encoded protein sequence recognize specifically MAP2a in rat brain homogenates. Moreover, although MAP2a, like MAP2b, is found in dendrites and cell bodies, its temporal appearance and cell type-specific distribution in rat brain differs from MAP2b.     

Microtubule-Associated Cyclic AMP-Dependent Protein Kinase in Drosophila melanogaster

Abstract: Microtubules were prepared from head extracts of the adult fruit fly, Drosophila melanogaster , by one-step, taxol-assisted polymerization. The microtubular fraction displayed cyclic AMP-dependent protein kinase (protein kinase A) activity, as witnessed by endogenous protein phosphorylation and by protein kinase assay. Microtubule-bound protein kinase A amounts to 4–5% of total soluble kinase activity, which is almost an order of magnitude less than in mammals. The high-molecular-weight microtu-bule-associated protein-2 (MAP-2), the main binding species for protein kinase A in mammalian brain microtubules, is not detectable in the fly system by protein staining and immunoblotting with anti-pig MAP-2 serum, as well as by hybridization of fly DNA with a cDNA probe for human MAP-2. Cyclic AMP removes a major part of the regulatory (R) subunit of the enzyme from Drosophila microtubules, as demonstrated by enzyme assay, autophosphoryla-tion of R subunit, and quantitating cyclic AMP binding sites. It is proposed that permanently elevated cyclic AMP levels may elute protein kinase A from crucial intracellular binding sites, thereby interfering with signal transduction.     

Characterization and Intracellular Distribution of Microtubule-Associated Protein 2 in Differentiating Human Neuroblastoma Cells

The use of a panel of monoclonal antibodies (mAbs) directed against different determinants of microtubule-associated protein 2 (MAP2) enabled us to identify two distinct high-molecular-mass MAP2 species (270 and 250 kDa) and a substantial amount of MAP2c (70 kDa) in human neuroblastoma cells. The 250-kDa MAP2 species appears to be confined to the human neuroblastoma cells and was not observed in microtubules (MTs) from bovine and rat brain, mouse neuroblastoma, or MTs from human cerebellum. A new overlay method was developed, which demonstrates binding of tubulin to human neuroblastoma high-molecular-mass MAP2 by exposing nitrocellulose-bound MT proteins under polymerization conditions to tubulin. Bound tubulin was detected with a mAb directed against beta-tubulin. The binding of tubulin to MAP2 could be abolished by a peptide homologous to positions 426-445 of the C-terminal region of beta-tubulin. Immunological cross-reactivity with several mAbs directed against bovine brain MAP2, taxol-promoted coassembly into MTs, and immunocytochemical visualization within cells were further criteria utilized to characterize these proteins as true MAPs. Indirect immunofluorescence with anti-MAP2 and anti-beta-tubulin mAbs demonstrated that there is a change in the spatial organization of MTs during induced cell differentiation, as indicated by the appearance of MT bundles and the redistribution of MAP2.     

Persistent poliovirus infection of human fetal brain cells.

It has been suggested that poliovirus (PV), the causative agent of poliomyelitis, could persist in surviving patients. We have previously shown that PV can persistently infect some human cell lines in vitro, particularly neuroblastoma cell lines. We report here an ex vivo model in which PV can persistently infect primary cultures of human fetal brain cells. Two mutations involving capsid residues 142 of VP2 and 95 of VP1 were repeatedly selected during the persistent infections. These residues are located in capsid regions known to be involved in interactions between PV and its receptor. During the first week after infection, viral antigens were found in cells of both the neuronal and glial lineages. In contrast, 2 weeks after infection, viral antigens were detected almost exclusively in cells of the neuronal lineage. They were detected predominantly in cells expressing a marker of early commitment to the neuronal lineage, MAP-5, particularly in neuroblasts. Viral antigens were also found in immature progenitors expressing a neuroepithelium marker, nestin, and in cells expressing a marker of postmitotic neurons, MAP-2. The presence of viral antigens in postmitotic neurons suggests that PV can persist in neurons of patients who have survived poliomyelitis.     

Reduction in Microtubule Dynamics In Vitro by Brain Microtubule-Associated Proteins and by a Microtubule-Associated Protein-2 Second Repeated Sequence Analogue

Abstract: Microtubule-associated protein (MAP) binding to assembled microtubules (MTs) can be reduced by the addition of polyglutamate without significant MT depolymerization or interference with MT elongation reactions. Ensuing polymer length redistribution in MAP-depleted MTs occurs on a time scale characteristic of that observed with MAP-free MTs. The redistribution phase occurs even in the absence of mechanical shearing and without appreciable effects from end-to-end annealing, as indicated by the time course of incremental changes in polymer length and MT number concentration. We also observed higher rates of MT length redistribution when the [MAP]/[tubulin] ratio was decreased. Together, these results demonstrate that MT length redistribution rates are greatly influenced by MAP content, and the data are compatible with the dynamic instability model. We also found that a peptide analogue corresponding to the second repeated sequence in the MT-binding region of MAP-2 can also markedly retard MT length redistribution kinetics, a finding that accords with the ability of this peptide to promote tubulin polymerization in the absence of MAPs and to displace MAP-2 from MTs. These results provide further evidence that MAPs can modulate MT assembly/disassembly dynamics and that peptide analogues can mimic the action of intact MAPs without the need for three contiguous repeated sequences in the MT-binding region.     

Association of Gap-43 (neuromodulin) with microtubule-associated protein MAP-2 in neuronal cells

Gap-43 (B-50, neuromodulin) is a presynaptic protein implicated in axonal growth, neuronal differentiation, plasticity, and regeneration. Its activities are regulated by its dynamic interactions with various neuronal proteins, including actin and brain spectrin. Recently we have shown that Gap-43 co-localizes with an axonal protein DPYSL-3 in primary cortical neurons. In the present study we provide evidence that Gap-43 co-localizes and potentially interacts with microtubule-associated protein MAP-2 in adult and fetal rat brain, as well as in primary neuronal cultures. Our studies suggest that this interaction may be developmentally regulated.