Levels Of Microtubule-associated Protein 2 Research Articles

Microtubule-associated protein 2 (MAP2) associates with the NMDA receptor and is spatially redistributed within rat hippocampal neurons after oxygen-glucose deprivation

MAP2 (microtubule-associated protein 2) is a cytoskeletal phosphoprotein that regulates the dynamic assembly characteristics of microtubules and appears to provide scaffolding for organelle distribution into the dendrites and for the localization of signal transduction apparatus in dendrites, particularly near spines. MAP2 is degraded after ischemia and other metabolic insults, but the time course and initial triggers of that breakdown are not fully understood. This study determined that MAP2 resides in a complex with the NMDA receptor, suggesting that spatially localized changes may be important in the mechanism of MAP2 redistribution and breakdown after oxygen-glucose deprivation (OGD). Using OGD in the adult rat hippocampal slice as a model system, this study demonstrated that MAP2 breakdown occurs very early after OGD, with the first statistical decrease in MAP2 levels within the first 30 min after the insult. There is a dramatic redistribution of MAP2 to the somata of pyramidal neurons, particularly neurons at the CA1-subiculum border. Free radicals and nitric oxide are not involved in the damage to MAP2. NMDA-receptor activation plays a prominent role in the MAP2 breakdown. In direct response to NMDA receptor activation, calcium influx, likely through the receptor ion channel complex, as well as release of calcium from the mitochondria through activation of the 2Na +–Ca 2+ exchanger of mitochondria, triggers MAP2 degradation. The proteolysis of MAP2 is limited by endogenous calpain activity, likely via the spatial access of calpain to MAP2.

Early Changes in MAP2 Protein in the Rat Hippocampus Following Soman Intoxication

We investigated the time course of both MAP2 (microtubule-associated protein 2) levels and its phosphorylation degree in the rat hippocampus during the first 90 min following the onset of soman-induced seizures. The quantitative immunoblot analysis of hippocampal extracts revealed that MAP2 increased significantly in response to a sustained epileptic activity (from 60 min of seizure duration). In addition, intense MAP2 dephosphorylation was also observed 60 to 90 min after the onset of seizures. The possible neuropathological consequences of these two early MAP2 changes are discussed in relation to the both excessive stimulation of glutamate receptors and subsequent dendritic spine alterations occuring in hippocampal neurons soon after soman intoxication.

Antipsychotic treatment induces alterations in dendrite- and spine-associated proteins in dopamine-rich areas of the primate cerebral cortex

Background: Mounting evidence indicates that long-term treatment with antipsychotic medications can alter the morphology and connectivity of cellular processes in the cerebral cortex. The cytoskeleton plays an essential role in the maintenance of cellular morphology and is subject to regulation by intracellular pathways associated with neurotransmitter receptors targeted by antipsychotic drugs. Methods: We have examined whether chronic treatment with the antipsychotic drug haloperidol interferes with phosphorylation state and tissue levels of a major dendritic cytoskeleton–stabilizing agent, microtubule-associated protein 2 (MAP2), as well as levels of the dendritic spine–associated protein spinophilin and the synaptic vesicle–associated protein synaptophysin in various regions of the cerebral cortex of rhesus monkeys. Results: Among the cortical areas examined, the prefrontal, orbital, cingulate, motor, and entorhinal cortices displayed significant decreases in levels of spinophilin, and with the exception of the motor cortex, each of these regions also exhibited increases in the phosphorylation of MAP2. No changes were observed in either spinophilin levels or MAP2 phosphorylation in the primary visual cortex. Also, no statistically significant changes were found in tissue levels of MAP2 or synaptophysin in any of the cortical regions examined. Conclusions: Our findings demonstrate that long-term haloperidol exposure alters neuronal cytoskeleton– and spine–associated proteins, particularly in dopamine-rich regions of the primate cerebral cortex, many of which have been implicated in the psychopathology of schizophrenia. The ability of haloperidol to regulate cytoskeletal proteins should be considered in evaluating the mechanisms of both its palliative actions and its side effects.

A brain derived peptide preparation reduces the translation dependent loss of a cytoskeletal protein in primary cultured chicken neurons.

Neuronal cytoskeletal proteins like the microtubule associated protein 2 (MAP2) are objected to pathological proteolysis in case of Alzheimer's disease and brain ischemia. The neurotrophic peptidergic drug Cerebrolysin (EBEWE Arzneimittel, Austria, Europe) is produced by a standardized enzymatic break-down of lipid free porcine brain proteins. Cerebolysin protected MAP2 in primary neuronal cultures after a brief histotoxic hypoxia and in a rat model of acute brain ischemia. Furthermore the drug was shown to inhibit the proteases mu- and m-calpain dose dependently in several cell free protease activity assays. The question if the higher MAP2 levels are due to an alleviation of proteolysis, to a higher synthesis rate or both is addressed in the current investigation: Monitoring the MAP2 content of primary neuronal cell cultures over a period of eight days revealed MAP2 to reach a peak level on day six in vitro followed by a degradation phase. In other experiments the protein synthesis of Cerebrolysin treated and untreated cells was blocked with cycloheximide at that moment when all cells exhibited the same MAP2 content. After the following MAP2 degradation phase--i.e. after eight days in vitro--the MAP2 contents were determined by western blotting. Cerebrolysin treated cells contained more MAP2 than untreated controls proving that the drug protects MAP2 independently from de novo synthesis, although further work is in progress to investigate if the drug supplementary boosts this effect by increasing MAP2 synthesis.

Isolation of a Potential Neural Stem Cell Line from the Internal Capsule of an Adult Transgenic Rat Brain

A thermosensitive mutation of simian virus 40 large T antigen (LTA) gene, the tsA58 gene, was cloned downstream of the 6-kbp neurofilament light chain promoter in pPOLYIII and injected into the pronucleus of fertilised oocytes of Sprague-Dawley rats to develop a strain harbouring six copies of the transgene. Immunocytochemical staining of hemizygous adult tissues with antibodies to the C-terminus of LTA showed that the inactive form of LTA was expressed only in the fibres of the internal capsule and in the choroid plexus of the brain. Culturing the former region at 33 degrees C, the permissive temperature for LTA, yielded a cell line, NF2C, which produced active LTA and grew at 33 degrees C but which produced only inactive LTA and eventually died at the non-permissive temperature of 39 degrees C. This clonal cell line was heterogeneous at 33 degrees C, producing the precursor neuronal cell marker nestin and the glial-specific markers glial fibrillary acidic protein, vimentin and S100A1, as well as weakly producing the neuronal cell markers 68-kDa neurofilament protein (NF68) and microtubule-associated protein 2 (MAP2) in different subpopulations of cells. However, at 39 degrees C, the cells produced dendritic, neuronal-like processes and elevated levels of NF68 and MAP2, as well as the neuronal markers synaptophysin, neurone-specific enolase, and low levels of tau, all determined by western blotting and immunofluorescent staining. Basic fibroblast growth factor enhanced the growth of the cells at 33 degrees C but also enhanced the formation of dendritic neuronal-like processes at 39 degrees C. It is suggested that NF2C represents a potential stem cell line from adult brain that expresses precursor and glial cell markers at 33 degrees C but undergoes partial differentiation to a neuronal cell phenotype at 39 degrees C.

Competing processes of cell death and recovery of function following ischemic preconditioning

The goal of the present study was to determine the neuroprotective efficacy of ischemic preconditioning using behavioral, electrophysiological and histological endpoints at various time points up to 90 days postischemia. Gerbils were exposed to a brief, non-injurious episode of forebrain ischemia (1.5 min) on each of 2 consecutive days. Three days following this preconditioning procedure, the animals received a 5 min occlusion. Other animals underwent sham surgery or a 5 min occlusion without preconditioning. Ischemic preconditioning appeared to provide striking histological protection at both rostral (∼80% and ∼67% of sham) and posterior levels of hippocampus (∼94% and ∼78% of sham) at 3 and 10 days survival, respectively. However, in spite of the near normal number of CA1 neurons, animals displayed marked impairments in an open field test of habituation as well as reduced dendritic field potentials in the CA1 area. Additionally, in ischemic animals the basal and apical dendritic regions of CA1 were nearly devoid of the cytoskeletal protein microtubule associated protein 2 (MAP2). Staining levels of MAP2 in preconditioned and sham animals were similar. With increasing survival time, open field behavior as well as CA1 field potential amplitude recovered. Nonetheless, CA1 cell death in ischemic preconditioned animals continued over the 90-day survival period ( P<0.05, vs. sham levels). Ischemic preconditioning provides a significant degree of neuroprotection characterized by a complex interplay of protracted cell death and neuroplasticity (recovery of function). These competing processes are best elucidated using a combination of functional and histological endpoints as well as multiple and extended survival times (i.e., greater than 7–10 days).

Rapid calpain I activation and cytoskeletal protein degradation following traumatic spinal cord injury: attenuation with riluzole pretreatment.

Immunocytochemical and immunoblotting techniques were used to investigate calpain I activation and the stability of the calpain-sensitive cytoskeletal proteins microtubule-associated protein 2 (MAP2) and spectrin at 1, 4, and 24 h after contusion injury to the spinal cord. Spinal cord injury resulted in the activation of calpain I at all time points examined, with the highest level of activation occurring at 1 h. At the same early time point, there was a loss of dendritic MAP2 staining in spinal cord sections, accompanied by pronounced perikaryal accumulation. The loss in MAP2 staining in the injured spinal cord progressed over the 24-h survival period to affect regions 3 mm distant to the site of injury. The presence of calpain I-specific spectrin degradation was apparent in neuronal cell bodies and fibers as early as 1 h after injury, with the most intense staining occurring within and juxtaposed to the injury site. Spectrin breakdown products in neuronal cell bodies declined rapidly at 4 h and were nearly undetectable at 24 h after injury. Immunoblot studies confirmed the immunocytochemical results by demonstrating a significant increase in calpain I activation, a significant decrease in MAP2 levels, and a significant increase in spectrin breakdown. Finally, treatment of animals with riluzole, an inhibitor of glutamate release, before surgery reduced significantly the loss of MAP2 levels observed at 24 h after injury. These results demonstrate that Ca2+-dependent protease activation and degradation of critical cytoskeletal proteins are early events after spinal cord injury and that treatments that minimize the actions of glutamate may limit their breakdown.

Secretion of amyloid β-protein by a mouse neuronal cell line : W. Araki, T. Kunishita, K. Takahashi, S. Ikeda and T. Tabira. Division of Demyelinating Disease and Aging, National Institute of Neuroscience, NCNP, Tokyo 187 Japan

Corticosterone, a principal glucocorticoid synthesized in the rodent adrenal cortex and secreted in response to stress, is reported to produce a biphasic effect on animal behavior. In this study, we determine that corticosterone administration produced different effects on depression-like behavior in mice depending on the length of time it was administered. In addition, we explored the indirect evidence at the cellular and molecular levels in order to support above assertion. Male mice received repeated injections of the vehicle and 20 mg/kg of corticosterone for 6, 18, and 36 days before being subjected to the forced swimming and tail suspension tests. After behavioral tests, we analyzed the number of neuron-specific nuclear protein (NeuN)-positive cells in the hippocampus, and the levels of two important cytoskeleton proteins, microtubule-associated protein 2 (MAP2) and neurofilament light chain protein (NF-L). Our results showed that 18-day and 36-day corticosterone injections caused increased depression-like behavior in male mice and significantly reduced the NF-L protein levels in the hippocampal tissues. However, 6-day corticosterone injection exhibited an anti-depressant effect accompanied by increased levels of MAP2 and NF-L in the hippocampus. Interestingly, no decrement was observed in NeuN-positive cells in the entire hippocampus throughout the experiments. The results support the view that short-term and long-term corticosterone administration produce opposite effects on depression-like behavior. Furthermore, the biphasic regulation of cytoskeleton proteins in the hippocampus might be a mechanism by which corticosterone treatments influence animal behavior.

Distinct mode of microtubule-associated protein 2 expression in the neuroblastoma/glioma cell line 108CC15/NG108-15.

The properties of microtubule-associated protein-2 (MAP-2) expression were examined in a transformed cell line, and compared to neurons from rodent brain where evidence supports both transcriptional and nontranscriptional regulation of MAP-2 synthesis. A monoclonal antibody that recognizes a common epitope in the adult (HMW MAP-2) and juvenile (MAP-2c) forms was used in an immunoblotting assay to assess the protein levels in actively dividing and differentiated neuroblastoma/glioma (108CC15, also designated NG108-15) cells. Multiply-phosphorylated MAP-2c was the predominant form in actively dividing cells, whereas HMW MAP-2 predominated in differentiated cells, which exhibited several other neuronal-like properties. A progressive increase in the levels of immunoreactive HMW MAP-2 was observed with increasing days of cell differentiation using dBcAMP as the inducing agent. However, the absolute levels of both HMW MAP-2 and MAP-2c in NG108-15 cells were significantly lower (at least 10-fold) than levels measured in rodent brain. To assess whether there are correspondingly lower levels of HMW MAP-2 and MAP-2c mRNAs in NG108-15 cells, relative to rodent brain, a highly sensitive RNA amplification assay (reverse transcription-polymerase chain reaction; RT-PCR) was developed. Oligonucleotide primers were designed to specify either HMW MAP-2 mRNA or MAP-2c mRNA, and whole tissue RNA extracted from adult and neonatal rodent brain was used to verify the reliability of the RT-PCR assay. Accordingly, PCR products of the predicted size, specificity, and abundance were obtained, with similar levels of HMW MAP-2 mRNA and proportionately higher levels of MAP-2c mRNA in neonatal brain, relative to adult brain. MAP-2c mRNA was the predominant transcript in actively dividing NG108-15 cells, and the amount of HMW MAP-2 mRNA gradually increased and became the predominant transcript in cells exposed to dBcAMP for 6-9 days. Thus, the observed changes in MAP-2-specific mRNAs during differentiation paralleled changes in expressed protein, suggesting that MAP-2 synthesis in NG108-15 cells is transcriptionally controlled. However, the levels of both MAP-2 mRNAs in NG108-15 cells were comparable to levels in rodent brain, despite the fact that MAP-2 protein levels are at least 10-fold lower in NG108-15 cells. These data suggest that the low levels of HMW MAP-2 and MAP-2c protein expression in NG108-15 cells are not due to correspondingly lower levels of MAP-2 mRNAs, and that transformed neuronal cell lines demonstrate a unique mode of MAP-2 regulation.

Decrease in amyloid precursor protein precedes hippocampal degeneration in rat brain following transient global ischemia

We have studied amyloid precursor protein (APP) expression in rat brain following transient global ischemia. Ischemic damage 24 h after 30 min of four-vessel occlusion (4VO) was limited to the caudate nucleus; hippocampal pyramidal neurons appeared histologically normal by light microscopy. Consistent with ongoing neurodegeneration in the caudate nucleus, microtubule-associated protein-2 (MAP-2) levels assessed by immunoblots were significantly reduced in homogenates of caudate nucleus after 4VO. MAP-2 levels In the hippocampus were comparable to control values. In contrast, full lenght APP levels in both the caudate nucleus and hippocampal homogenates were significantly decreased following 4VO despite normal hippocampal morphology at 24 h. These findings suggest that decrements in full length APP precede overt neuronal damage and may play a role in the subsequent delayed neurodegeneration in the hippocampus.

Microtubule-associated protein 2 levels decrease in hippocampus following traumatic brain injury.

We examined microtubule-associated protein 2 (MAP2) levels in hippocampal and cortical tissue 3 h following moderate traumatic brain injury (TBI) in the rat. MAP2 levels were assayed by quantitative immunoreactivity in tissue fractions obtained from naive, sham-injured, or fluid percussion-injured animals. Tissues were homogenized in the presence of protease inhibitors (0.3 mM phenylmethylsulfonyl fluoride, PMSF), a specific calpain inhibitors (0.1 mM leupeptin), and chelators (2 mM ethylene glycol-bis-tetraacetic acid, EGTA; 1 mM ethylenedinitrilo-tetraacetic acid, EDTA) to eliminate in vitro MAP2 proteolysis during tissue processing. Compared to naive rats, sham injury had no effect on soluble MAP2 levels in either cortex (105.0 +/- 4.4% of naive value) or hippocampus (106.6 +/- 5.2% of naive value). However, TBI caused a significant (p < 0.005) decrease in hippocampal MAP2 levels (55.7 +/- 5.9% of sham-injured controls). The effect appeared to be regionally selective, since the MAP2 decrease did not occur in cortex (89.1 +/- 1.4%). The degree of MAP2 decrease in hippocampus was similar in both membrane (57.8%) and cytosolic (55.7%) fractions, ruling out the possibility of partitioning artifacts. The data suggest that sublethal alterations of neuronal structure and function caused by MAP2 degradation may play an important role in the development of TBI-induced functional deficits. Since MAP2 is exclusively associated with the cytoskeleton in somal and dendritic compartments of neurons, the pathophysiology of sublethal magnitudes of TBI may also involve dendritic and somal dysfunction.

Regulation of microtubule associated protein 2 (MAP2) expression by nerve growth factor in PC12 cells

In the presence of nerve growth factor (NGF), PC12 cells cease to divide and differentiate, extending long microtubule-containing neurites. We showed by immunoblot analysis that MAP2 was detectable in PC12 after 4 days of NGF treatment and that its levels increased five- to sevenfold after 12 days of NGF treatment. The apparent molecular weight of MAP2 in PC12 cells was similar to that of rat brain MAP2 (280,000), with a doublet representing the MAP2 isoforms. However, the relative levels of MAP2 in differentiated PC12 cells were 5–10% of those found in rat brain. Immunofluorescence analysis of NGF-treated PC12 cells revealed that MAP2 co-localized with tubulin and was present in cell bodies and neurites. Northern blot analysis showed that the levels of MAP2 mRNA increased in PC12 cells during NGF-treatment in a pattern that paralleled the protein levels, suggesting that MAP2 expression is transcriptionally regulated.

Suppressive effect of E-64c on ischemic degradation of cerebral proteins following occlusion of the middle cerebral artery in rats

Microtubule-associated protein 2 (MAP2) levels in the left cerebral hemisphere decreased significantly 3 days after occlusion of the left middle cerebral artery in rats to29 ± 16.3% of control levels. Since MAP2 is one of the substrates of calpain, E-64c, a synthetic calpain inhibitor, was administered at a dose of 400 mg/kg twice a day for 3 days, with the first dose being given before the production of ischemia. This depletion was significantly inhibited in vivo by E-64c ( P < 0.05) to increase MAP2 levels to55 ± 25.7% of control levels. E-64c had no significant effect on the ischemia-induced depletion of myelin-associated glycoprotein. Sham-operated rats were used as controls. Our results suggest that calpain is partially involved in the degradation of MAP2, and that the use of calpain inhibitors can be a useful clinical approach to cerebral ischemia.

Developmental regulation of microtubule-associated protein 2 expression in regions of mouse brain.

The relative levels of microtubule-associated protein 2(MAP2) were determined during postnatal development of the mouse in six different discrete brain regions: cerebellum, cortex, hippocampus, olfactory bulb, brainstem, and hypothalamus. Brain homogenates were electrophoresed on sodium dodecyl sulfate-containing gels and analyzed by immunoblotting with MAP2-specific antibodies. The levels of MAP2 in each region were determined using radiolabeled secondary antibodies and densitometric quantification of the autoradiograms over a range that was determined to have a linear response. The results indicated that in all regions and at all ages there was only one high-molecular-weight polypeptide of MAP2, which did not change in electrophoretic mobility after dephosphorylation. In most regions, the levels of MAP2 increased during the first 2 postnatal weeks. However, there were differences in the time course and relative levels of MAP2 between regions. In addition, all regions of the brain expressed the low-molecular-weight form of MAP2 (MAP2c) that was present at birth as a heterogeneous group of polypeptides with an apparent molecular weight of 70K. Most of the heterogeneity of MAP2c, however, was eliminated after dephosphorylation. The levels of MAP2c decreased dramatically after 2 weeks postnatally, except for the olfactory bulb, where the levels of MAP2c remained relatively high even in adults.

Regulation of microtubule-associated protein 2 (MAP2) mRNA expression during rat brain development

The expression of MAP2 during rat brain development was studied by using specific antibodies and cDNA probes. MAP2 cDNAs were isolated from a rat brain lambda gt11 library, and their identity was confirmed by the reactivity of their fusion proteins with several independent monoclonal antibodies that recognize MAP2. Northern blot analyses of the RNA prepared from whole brains, cerebral cortex, hypothalamus, brain stem, olfactory bulbs, and cerebellum showed that the levels of MAP2 mRNA increase during the initial phase of development, reach a maximum between postnatal weeks 2 and 3, and then decrease in the adult. The time course and the kinetics of this change varied between different brain regions and appeared to reflect the pattern of morphological changes in these regions. RNA blots were also analyzed with beta-tubulin and beta-actin cDNA probes to ensure the quality and the quantity of the RNA. The levels of MAP2 mRNA and protein showed similar changes during the initial part of brain development and suggested a transcriptional control. However, while MAP2 protein levels remained high throughout development, MAP2 mRNA levels decreased in adulthood. We suggest that the increased stability of the MAP2 molecule may be a contributing factor in the developmental regulation of steady-state levels of MAP2.

Ontogenesis of microtubule-associated protein 2 (MAP2) in embryonic mouse cortex

The developing neocortex in mice from embryonic day 13 (E13) until birth (E19) was immunoreacted with a monoclonal antibody for microtubule-associated protein 2 (MAP2) that is highly specific for neuronal somata and dendrites. In E13 neocortex there was no detectable MAP2 immunoreactivity on tissue sections or on gel blots. From E14 to birth the MAP2 immunoreactivity was present in both tissue sections and immunoblots of homogenized cortex. In the neocortex the staining pattern was lamina-specific. The molecular layer and the cortical subplate contained the most dense staining of dendrites and cell somata. The cortical plate showed weak to moderate staining at these ages while the intermediate and ventricular zones were not stained above background control levels. Gel blots correspondingly did not show detectable levels of MAP2 until E14. Ultrastructural data suggest that MAP2 is present in dendrites in each of the laminae. The laminar pattern of MAP2 immunoreactivity may be due to either the higher density of differentiating dendrites in the molecular and subplate layers or to compartmentalization of MAP2 within individual cortical neurons.