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.     

The 72-kDa Microtubule-Associated Protein from Porcine Brain

A microtubule-associated protein (MAP) with a molecular mass of 72-kDa that was purified from porcine brain by using its property of heat stability in a low pH buffer was characterized. Low-angle rotary shadowing revealed that the 72-kDa protein was a rodlike protein approximately 55-75 nm long. The 72-kDa protein bound to microtubules polymerized from phosphocellulose column-purified tubulin (PC-tubulin) with taxol and promoted the polymerization of PC-tubulin in the absence of taxol. Microtubules polymerized by the 72-kDa protein showed a tendency to form bundles of several microtubules. Quick-freeze, deep-etch electron microscopy revealed that the 72-kDa protein formed short crossbridges between microtubules. We performed peptide mapping to analyze the relationship of the 72-kDa protein to other heat-stable MAPs, and the results showed some resemblance of the 72-kDa protein to MAP2. Cross-reactivity with a monoclonal anti-MAP2 antibody further suggested that the 72-kDa protein and MAP2 are immunologically related. To study the relationship between the 72-kDa protein and MAP2C, a smaller molecular form of MAP2 identified in juvenile rat brain, we prepared the 72-kDa protein from rat brain by the same method as that used for porcine brain. The fact that the 72-kDa protein from juvenile rat brain was also stained with our monoclonal anti-MAP2 antibody also suggested that the 72-kDa protein is an MAP2C homologue of the porcine brain.     

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.     

NMDA and Nitric Oxide Increase Microtubule-Associated Protein 2 Gene Expression in Hippocampal Granule Cells

Abstract: Microtubule-associated protein 2 (MAP2), a component of the neuronal cytoskeleton, has attracted attention as a possible cellular substrate linking hippocampal N -methyl- d -aspartate (NMDA) receptor stimulation to alterations in cellular morphology. We show here that microinjection of NMDA, 8-bromo-cyclic GMP, or sin-1 molsidomine (which spontaneously releases nitric oxide), onto the molecular layer of the hippocampal dentate gyrus, increased the levels of MAP2 mRNA in the affected granule cells. No changes were observed in the levels of mRNAs encoding several other cytoskeletal components. This shows that hippocampal NMDA receptor stimulation can potentially initiate a long-term alteration in dendritic structure by affecting MAP2 gene expression and provides the first evidence that nitric oxide release in vivo, acting through cyclic GMP-dependent protein kinase, can cause long-term changes in neuronal function by modulating gene expression.     

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.     

Tyrosine Phosphorylation of Microtubule-Associated Protein Kinase After Transient Ischemia in the Gerbil Brain

The tyrosine phosphorylation of microtubule-associated protein (MAP) kinase was examined in the gerbil brain after transient ischemia and reperfusion. Phosphorylation of MAP kinase was maximal within 1 min of reperfusion following 5 min of ischemia and returned to control levels as early as 5 min postischemia. The greatest increase in MAP kinase phosphorylation was detected in the hippocampus, with minor increases in other ischemic regions of the brain. Several tyrosine-phosphorylated proteins were detected in the gerbil hippocampus; however, the ischemia and reperfusion injury only increased tyrosine phosphorylation of MAP kinase. The increase in tyrosine phosphorylation was prevented by the N-methyl-D-aspartate (NMDA) receptor blocker (+)-MK-801, whereas a non-NMDA receptor blocker, 6-cyano-7-nitroquinoxaline-2,3-dione, was ineffective. Pretreatment of gerbils with calcium channel blockers also prevented the tyrosine phosphorylation of MAP kinase in the ischemic brain. Altogether, these results imply an involvement of glutamate receptors and calcium during the tyrosine phosphorylation of MAP kinase. Tyrosine phosphorylation was also prevented when ischemia and reperfusion were conducted under hypothermic conditions, which protect against neurodegenerative damage. These findings implicate a role for MAP kinase in neuronal damage resulting from ischemia and reperfusion.     

Ca2+– and Calmodulin-Dependent Phosphorylation of Microtubule-Associated Protein 2 and t Factor, and Inhibition of Microtubule Assembly

Microtubule-associated proteins (MAPs) were phosphorylated by a Ca2+- and calmodulin-dependent protein kinase from rat brain cytosol. The maximal amount of phosphate incorporated into MAPs was 25 nmol of phosphate/mg protein. A Ka value of the enzyme for calmodulin was 57.0 nM, with MAPs as substrates. Among MAPs, MAP2 and tau factor were phosphorylated in a Ca2+- and calmodulin-dependent manner. The phosphorylation of MAPs led to an inhibition of microtubule assembly in accordance with its degree. This reaction was dependent on addition of the enzyme, Ca2+, and calmodulin, and had a greater effect on the initial rate of microtubule assembly rather than on the final extent. The critical tubulin concentration for microtubule assembly was unchanged by the MAPs phosphorylation. Therefore assembly and disassembly of brain microtubule are regulated by the Ca2+- and calmodulin-dependent protein kinase that requires only a nanomolar concentration of calmodulin for activation.     

Developmental Expression of the Glycine Transporters GLYT1 and GLYT2 in Mouse Brain

Abstract: Using immunocytochemical localization, the distribution of the glycine transporters GLYT1 and GLYT2 in the developing mouse brain was studied. GLYT1 and GLYT2 immunoreactivity begins during the period of fiber outgrow and synaptogenesis. GLYT2 is first expressed in spinal and spinothalamic white matter and is followed by the expression of synaptophysin. In the postnatal stages, GLYT2 staining in the white matter disappears, and a punctuated pattern in the gray matter emerges. In contrast, in the fetal brain GLYT1 immunoreactivity coincides with gray matter neuropil and processes of radial glia. GLYT1 is distributed over a much wider area of the brain than GLYT2. However, the distribution of these two GLYTs implies that GLYT1 and GLYT2 operate in concert within the area where both are present. At the day 12 embryo stage, GLYT1 antibodies stain the liver, and later they also react with the pancreas and the gastroduodenal junction. No other organs exhibit significant GLYT1 immunoreactivity. We additionally observed the presence of GLYT1 in rat fetal cerebral cortex and hippocampus, which was not detected in fetal mouse brain. Moreover, GLYT1 immunoreactivity was found in the mouse floor plate and the ventral commissure but was not present in the same regions in rats. These findings suggest possible differences in the expression of GLYT1 between these two species.     

A 70-Kilodalton Microtubule-Associated Protein (MAP2c), Related to MAP2

Microtubule-associated protein 2 (MAP2) from adult brain consists of a pair of high molecular mass (280 kilodaltons) polypeptides, MAP2a and MAP2b. Juvenile brain microtubules also contain a 70-kilodalton protein that cross-reacts with monoclonal antibodies against these high molecular weight MAP2s. We have analyzed the relationship between this 70-kilodalton protein and MAP2 by peptide mapping. Our results show that the 70-kilodalton species bears strong homology to the MAP2 molecules and that it is distinct from the tau MAPs. We propose the name MAP2c for this low molecular weight MAP2 species. MAP2c is developmentally regulated in brain, being more abundant in neonatal tissue than in the adult. In several cell lines, MAP2c is the sole MAP2 species expressed. We examined homogenates from both juvenile brain and MAP2c-containing cell lines for evidence of a protease activity that might be responsible for generating MAP2c from either MAP2a or MAP2b. No such activity was found, suggesting that MAP2c is an independently synthesized MAP2 species some 200 kilodaltons smaller than the previously recognized forms.     

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.     

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.     

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.     

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.     

Expression of Microtubule-Associated Proteins During the Early Stages of Neurite Extension by Brain Neurons Cultured in a Defined Medium

Immunoblotting analysis was used to identify the microtubule-associated proteins (MAPs) present in cultures of mouse brain neurons. Polyclonal antibodies were raised against the two main adult brain MAPs, i.e., MAP2 (300 kDa) and tau (60-70 kDa). Whatever the stage of the culture, which was performed in a defined medium (3 or 6 days), the anti-MAP2 serum detected several high-molecular-weight components (including MAP2) and an entity with 62-65 kDa. Anti-tau revealed essentially a major peak of 48 kDa (young tau) but also slightly cross-reacted with the 62-65 kDa entity. During the culture period (0-6 days) the cells developed progressively a dense neuritic network; the concentration of the different MAPs increased in parallel but at different rates depending on the different species. The increase in concentration of the high-molecular-weight components occurred before that of 48-kDa tau. This suggests that high-molecular-weight MAPs and 48-kDa tau might be involved respectively in the initiation and elongation of neurites. In contrast, and since the main developmental changes in tau composition seen in vivo did not occur during the time course of the culture, this transition might be related to later events of neuronal differentiation.     

Developmental Expression of the Outer Hair Cell Motor Prestin in the Mouse

The development of motor protein activity in the lateral membrane of the mouse outer hair cell (OHC) from postnatal day 5 (P5) to P18 was investigated under whole-cell voltage clamp. Voltage-dependent, nonlinear capacitance (C _v), which represents the conformational fluctuations of the motor molecule, progressively increased during development. At P12, the onset of hearing in the mouse, C _v was about 70% of the mature level. C _v saturated at P18 when hearing shows full maturation. On the other hand, C _lin, which represents the membrane area of the OHC, showed a relatively small increase with development, reaching steady state at P10. This early maturation of linear capacitance is further supported by morphological estimates of surface area during development. These results, in light of recent prestin knockout experiments and our results with quantitative polymerase chain reaction, suggest that, rather than the incorporation of new motors into the lateral membrane after P10, molecular motors mature to augment nonlinear capacitance. Thus, current estimates of motor protein density based on charge movement may be exaggerated. A corresponding indicator of motor maturation, the motor’s operating voltage midpoint, V _pkcm, tended to shift to depolarized potentials during postnatal development, although it was unstable prior to P10. However, after P14, V _pkcm reached a steady-state level near −67 mV, suggesting that intrinsic membrane tension or intracellular chloride, each of which can modulate V _pkcm, may mature at P14. These developmental data significantly alter our understanding of the cellular mechanisms that control cochlear amplification and provide a foundation for future analysis of genetic modifications of mouse auditory development.     

In Vitro Phosphorylation of Microtubule-Associated Protein 2: Differential Effects of Cyclic AMP Analogues

Microtubules purified from brain tissue contain endogenous cyclic AMP (cAMP)-dependent protein kinase activity, and microtubule-associated protein 2 (MAP2) is the major substrate. Beef brain microtubules were prepared and used as a model system to study the differential effects of rationally selected cyclic nucleotide analogues on microtubule receptor protein kinase. Data are presented to indicate that the following molecular interactions are essential for activation of the phosphorylation of MAP2: (a) hydrogen bond formation toward the 2', 3', or 5' position, (b) interaction with phosphorus, and (c) no hydrogen bonds but hydrophobic interactions at the base moiety. Thus, the activation mechanism of the type II protein kinase associated with brain microtubules resembles the mechanism found in protein kinases of other systems. In addition, we have studied the effect of the two diastereomers of adenosine 3',5'-monophosphorothioate (cAMPS). The (Sp)-cAMPS isomer was found to activate MAP2 protein kinase, whereas the (Rp)-cAMPS isomer had no activating effect. In contrast, this compound was able to inhibit cAMP-stimulated MAP2 phosphorylation and thus acts as an antagonist of the Sp diastereomer and cAMP. Hence, this analogue provides a useful means to clarify further the effect of cAMP-dependent phosphorylation on functional properties in microtubules in general.     

Localization of the Phosphorylation Sites for Different Kinases in the Microtubule-Associated Protein MAP2

The phosphorylation of microtubule-associated protein 2 (MAP2) by four different kinases was studied in vitro to determine whether MAP2 is phosphorylated in its tubulin binding region or in the microtubule projection portion. Fragments corresponding to both regions of MAP2 were produced not only by chymotrypsin or trypsin digestion, but also using pepsin, a broad chain-specificity protease, a result supporting previous notions of the two-domain structure of MAP2. The position of these two functional domains was determined with respect to the carboxy terminal of the molecule, by labeling MAP2 exclusively at the carboxy terminal and subjecting it to pepsin digestion. The results suggested that the projection region of MAP2 contained the carboxy terminal of the protein. A phosphorylation map was constructed by subjecting phosphorylated MAP2 to enzymatic digestion using Staphylococcus aureus V8 protease or to chemical cleavage using N-chlorosuccinimide. The results indicated that all four kinases phosphorylated MAP2 in a 42-kilodalton peptide that contained the tubulin binding region but differed in the level and localization of the sites at which they phosphorylated the projection of MAP2.