High Molecular Weight Microtubule-associated Proteins Research Articles

Interaction between very‐KIND Ras guanine exchange factor and microtubule‐associated protein 2, and its role in dendrite growth – structure and function of the second kinase noncatalytic C‐lobe domain

The kinase noncatalytic C-lobe domain (KIND) is a putative protein-protein interaction module. Four KIND-containing proteins, Spir-2 (actin-nuclear factor), PTPN13 (protein tyrosine phosphatase), FRMPD2 (scaffold protein) and very-KIND (v-KIND) (brain-specific Ras guanine nucleotide exchange factor), have been identified to date. Uniquely, v-KIND has two KINDs (i.e. KIND1 and KIND2), whereas the other three proteins have only one. The functional role of KIND, however, remains unclear. We previously demonstrated that v-KIND interacts with the high-molecular weight microtubule-associated protein 2 (MAP2), a dendritic microtubule-associated protein, leading to negative regulation of neuronal dendrite growth. In the present study, we analyzed the structure-function relationships of the v-KIND-MAP2 interaction by generating a series of mutant constructs. The interaction with endogenous MAP2 in mouse cerebellar granule cells was specific to v-KIND KIND2, but not KIND1, and was not observed for the KINDs from other KIND-containing proteins. The binding core modules critical for the v-KIND-MAP2 interaction were defined within 32 residues of the mouse v-KIND KIND2 and 43 residues of the mouse MAP2 central domain. Three Leu residues at amino acid positions 461, 474 and 477 in the MAP2-binding core module of KIND2 contributed to the interaction. The MAP2-binding core module itself promoted dendrite branching as a dominant-negative regulator of v-KIND in hippocampal neurons. The results reported in the present study demonstrate the structural and functional determinant underlying the v-KIND-MAP2 interaction that controls dendrite arborization patterns.

HMWMAP2: New perspectives on a pathway to dendritic identity

Neuronal polarity is established by the differentiation of two types of cytoplasmic processes: dendrites and the axon. These processes can be distinguished by their composition in microtubule-associated proteins, the high molecular weight MAP2 proteins (HMWMAP2) being found in the dendrites and tau proteins in the axon. It is believed that the main contribution of HMWMAP2 to the acquisition and maintenance of dendrites is to promote microtubule assembly and stability. However, recent studies force us to enlarge our view on how HMWMAP2 might contribute to defining the role of the dendritic microtubules. The purpose of this article is to convey our view that HMWMAP2 are important players in defining the contribution of microtubules to dendritic identity by anchoring membranous organelles and signaling proteins to the dendritic microtubules and by being a receptor for neurosteroids.

Failure of Neuronal Maturation in Alzheimer Disease Dentate Gyrus

The dentate gyrus, an important anatomic structure of the hippocampal formation, is one of the major areas in which neurogenesis takes place in the adult mammalian brain. Neurogenesis in the dentate gyrus is thought to play an important role in hippocampus-dependent learning and memory. Neurogenesis has been reported to be increased in the dentate gyrus of patients with Alzheimer disease, but it is not known whether the newly generated neurons differentiate into mature neurons. In this study, the expression of the mature neuronal marker high molecular weight microtubule-associated protein (MAP) isoforms MAP2a and b was found to be dramatically decreased in Alzheimer disease dentate gyrus, as determined by immunohistochemistry and in situ hybridization. The total MAP2, including expression of the immature neuronal marker, the MAP2c isoform, was less affected. These findings suggest that newly generated neurons in Alzheimer disease dentate gyrus do not become mature neurons, although neuroproliferation is increased.

Changes in microtubule-associated protein-2 (MAP2) expression during development and after status epilepticus in the immature rat hippocampus

In this study, we analyzed the spatiotemporal expression patterns of the high-molecular weight (MAP2a and b) and low-molecular weight (MAP2c and d) cytoskeletal microtubule-associated protein-2 (MAP2) isoforms with Western blotting, and the cellular localization of the high-molecular weight MAP2 isoforms with immunocytochemistry in the hippocampi of 1- to 21-day-old rats. Moreover, the temporal profile (from 30min to 1 week) of MAP2 isoform reactivity to kainic acid-induced status epilepticus was studied in P9 rats. During development, the expression of the high-molecular weight MAP2 isoforms significantly increased, while the low-molecular weight isoforms decreased, the most prominent changes occurring during the second postnatal week. This developmental increase in the high-molecular weight MAP2 expression was also confirmed with immunocytochemistry, which showed increased immunoreactivity, particularly in the molecular layers of the dentate gyrus, and in CA1 and CA3 stratum radiatum. In 9-day-old rats, status epilepticus resulted in a rapid transient increase (about 210%) in the high-molecular weight MAP2 expression, without any effect on the low-molecular weight MAP2. Moreover, disturbed dendritic structure in the CA1 and CA3 stratum radiatum was manifested as formation of varicosities 3h after the kainic acid treatment. The strictly developmentally regulated MAP2 isoform expression suggests different functional roles for these proteins during the postnatal development in the rat hippocampus. Moreover, high-molecular weight MAP2s may play a role in nerve cell survival during cell stress.

Semaphorin 3C preserves survival and induces neuritogenesis of cerebellar granule neurons in culture.

Semaphorins (sema) constitute a family of molecules sharing a common extracellular domain (semaphorin domain). This family includes several types of secreted and membrane-associated molecules that are grouped into eight subclasses (subclasses 1-7 and viral semaphorins). Subclass 3 semaphorins are secreted molecules involved in axonal guidance, mainly through repulsive gradients and induction of growth cone collapse. More recently sema 3 molecules have been identified as positive factors in dependence of the type of neurons. Besides their axonal guidance function, some semaphorins have been implicated in apoptosis and survival. We investigated the effect of sema3C on survival and neurite outgrowth of rat cerebellar granule neurons (CGNs) in culture. 3T3 cells were stably transfected with sema3C. Several clonal lines were established and tested for their neuritogenic activity and one, S3C-8, was selected for the bulk of experiments. S3C-8 was co-cultured with CGNs. Sema3C enhanced CGN viability as assessed in co-cultures of CGNs with monolayers of S3C-8 in comparison with co-cultures of CGNs with control mock-transfected 3T3 cells. Moreover sema3C induced neuritogenesis of cultured CGNs, which express neuropilin-1 and -2. S3C-8 cells, overexpressing sema3C, were significantly more neuritogenic for CGN than poly l-lysine (PLL), a positive substrate for CGNs, as assessed by the measurement of the length of neurites and confirmed by Tau expression along the time of culture. CGNs co-cultured with S3C-8, showed up-regulation of the expression of axonal microtubule-associated proteins (MAPs) such as Tau, phosphorylated MAP2C and mode I-phosphorylated MAP1B compared with neurons cultured on control 3T3 cells. We also found increased expression of a specific marker of neuronal cell bodies and dendrites, high molecular weight MAP2 (HMW-MAP2). Interestingly, there was no accompanying up-regulation of a marker enriched within the neuronal somatodendritic domain, mode II-phosphorylated MAP1B. These data support the idea that secreted sema3C favors survival and neuritogenesis of cultured CGNs.

Regulation of phosphorylation of neuronal microtubule-associated proteins MAP1b and MAP2 by protein phosphatase-2A and -2B in rat brain

The function of the neuronal high molecular weight microtubule-associated proteins (MAPs) MAP1b and MAP2 is regulated by the degree of their phosphorylation, which in turn is controlled by the activities of protein kinases and protein phosphatases (PP). To investigate the role of PP in the regulation of the phosphorylation of MAP1b and MAP2, we used okadaic acid and cyclosporin A to selectively inhibit PP2A and PP2B activities, respectively, in metabolically competent rat brain slices. The alteration of the phosphorylation levels of MAP1b and MAP2 was examined by Western blots using several phosphorylation-dependent antibodies to these proteins. The inhibition of PP2A, and to a lesser extent of PP2B, was found to induce an increased phosphorylation of MAP1b and inhibit its microtubule binding activity. Immunocytochemically, a marked increase in neuronal staining in inhibitor-treated tissue was observed with antibodies to the phosphorylated MAP1b. The inhibition of PP2A but not of PP2B also induced phosphorylation of MAP2 at multiple sites and impaired its microtubule binding activity. These results suggest that PP2A might be the major PP that participates in regulation of the phosphorylation of MAP1b and MAP2 and their biological activities.

Multiple isoforms of the high molecular weight microtubule associated protein XMAP215 are expressed during development in Xenopus.

We have cloned and sequenced cDNAs encoding two isoforms of XMAP215, a high molecular weight microtubule-associated protein identified in Xenopus eggs. XMAP215 is approximately 80% identical in amino acid sequence to the product of ch-TOG, a cDNA that is over expressed in certain human tumors [Charrasse et al., 1995: Eur J Biochem 234:406-413]. Northern and Western blots demonstrated that XMAP215 is expressed throughout development, from oogenesis to tadpole. We identified two XMAP215 transcripts differing only in the presence of a 108-bp sequence encoding a 36 amino acid insert. RT-PCR revealed that the transcripts encoding these two isoforms are expressed at distinct times during development: a transcript containing the insert (encoding XMAP215(M)) is expressed during oogenesis and is present through gastrulation. The second transcript (encoding XMAP215(Z)) lacks the 108-bp insert and is expressed from gastrulation onward. In situ hybridization demonstrated that XMAP215 transcripts are localized to the ectoderm of early embryos and in the developing nervous system during later development. These results suggest that XMAP215 plays important roles in at least two phases of development: (1) regulating the assembly of MTs during the rapid cell divisions after fertilization, and (2) regulating MT assembly during the development of the nervous system.

Pollen tube taxol dependent structures co-assemble with neuronal HMW MAPs (MAP2)

Abstract Pollen tube microtubules (MTs) are as dynamic as animal MTs and they may interact with plasma membrane, endoplasmic reticulum, Golgi apparatus, mitocondria and a variety of cytoplasmic proteins. Bridges connecting MTs to each other and to membranes have been documented in pollen tubes by electron microscopy; however, the biochemical and molecular nature of these linkages is not known. In other cell types interaction between organelles and MTs require the participation of Microtubule-Associated Proteins (MAPs) that bridge the cytoskeleton to these organelles. Although biochemical documentation of such bridging MAPs in plant cells is lacking, it is reasonable to assume, by analogy with the animal systems, that specialized MAPs regulate MTs polymerization and dynamic in pollen tube. As a first step toward testing this hypothesis, the ability of Nicotiana tabacum pollen tube taxol-stabilized MTs to bind mammalian brain High Molecular Weight MAPs (HMWMAPs)(MAP2) was tested. This association analysis r...

Novel Low Molecular Weight Microtubule-associated Protein-2 Isoforms Contain a Functional Nuclear Localization Sequence

Known high and low molecular weight (LMW) MAP2 protein isoforms result from alternative splicing of the MAP2 gene. Contrary to previous reports that MAP2 is neural-specific, we recently identified MAP2 mRNA and protein in somatic and germ cells of rat testis, and showed the predominant testicular isoform is LMW. Although cytoplasmic in neural tissue, MAP2 appeared predominantly nuclear in germ cells using immunohistochemistry. We sought to determine whether this unexpected localization was due to the inclusion of exon 10 within novel LMW MAP2 isoforms. Normally excluded from the LMW MAP2c, exon 10 harbors a putative CcN motif, comprising a nuclear localization sequence (NLS) flanked by regulatory phosphorylation sites for protein kinase CK2 and cdc2 kinase. Characterization of MAP2 mRNA in adult and immature brain and testis, by reverse transcriptase-polymerase chain reaction/Southern analysis and Northern blot, identified novel LMW forms containing exons 10 and 11, previously detected only in high molecular weight MAP2a and 2b. The MAP2 NLS targeted a large heterologous protein to the nucleus, as demonstrated using bacterially expressed MAP2-CcN-beta-galactosidase fusion protein and an in vitro nuclear import assay. Antibodies raised against the fusion protein produced a testicular immunohistochemical staining pattern correlating with MAP2 protein distribution in the nucleus of most germ cells, and precipitated both approximately 70-kDa and >220-kDa proteins recognized by the commercial MAP2-specific HM2 monoclonal antibody, supporting our hypothesis of a novel LMW MAP2 isoform. These results demonstrate the presence of a functional NLS in MAP2 and indicate that novel LMW MAP2 isoforms may be targeted to the nucleus in both neural and non-neuronal tissues.

Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons

The phosphorylation state of cytoskeletal proteins, including certain microtubule-associated proteins, may influence the development and plasticity of axons and dendrites in mammalian neuron in response to appropriate extracellular stimuli. In particular, high molecular weight microtubule-associated protein 2, has been implicated in dendrite growth and synaptic plasticity and is thought to be modulated by phosphorylation and dephosphorylation. We have previously determined that threonines 1620/1623 on the microtubule-associated protein 2 molecule become phosphorylated in vivo and are targets for proline-directed protein kinases in vitro. Using the phosphorylated site-specific antibody 305, we now report the decreased phosphorylation state of high molecular weight microtubule-associated protein 2 during the development of cultured cerebellar granule neurons. Phosphorylation of high molecular weight microtubule-associated protein 2 at this site is significantly inhibited by lithium in short-term cultured neurons, which suggests the implication of glycogen synthase kinase-3. In long-term cultured neurons, it is also partially inhibited by PD098059, an inhibitor of extracellular signal-regulated protein kinase activation, which indicates an additional contribution of this kinase to high molecular weight microtubule-associated protein 2 phosphorylation at this stage. Both in short-term and long-term cultured neurons, okadaic acid augments high molecular weight microtubule-associated protein 2 phosphorylation at this site through the inhibition of protein phosphatases 1 and/or 2A. Finally, glutamate receptor activation leads to a dephosphorylation of high molecular weight microtubule-associated protein 2 at this site which can also be effectively prevented by okadaic acid. These results are consistent with the participation of glycogen synthase kinase-3, extracellular signal-regulated protein kinases and protein phosphatases 1 and 2A, in the regulation of microtubule-associated protein 2 phosphorylation within living neurons, which may be modulated by extracellular signals like the neurotransmitter glutamate.

Phosphorylation of microtubule-associated proteins from the ovaries of hemipteran insects by MPF and MAP kinase: possible roles in the regulation of microtubules during oogenesis.

Nutritive tubes that link the developing oocytes to the nurse cells in ovarioles of hemipteran insects contain extensive arrays of microtubules. These are established, then later depolymerised, by developmentally regulated processes. Breakdown of the microtubules corresponds with the activation of M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase), later in oogenesis, as the oocytes proceed to arrest at the first meiotic metaphase [Lane and Stebbings, Roux's Arch Dev Biol 205:150-159 (1995)]. The mechanisms that lead to the breakdown of nutritive tube microtubules are unknown. Here, we have investigated the possibility that the insect ovarian microtubules are regulated by MPF- or MAP kinase-dependent phosphorylation, focusing upon the prominent high molecular weight microtubule-associated protein (HMW MAP) enriched in this system, which is a potential target for protein kinase activity in vivo. We have purified the prominent HMW MAPs from the ovaries of two species of hemipterans, and have shown them to be substrates in vitro for the activities of MPF and MAP kinase. However, although the catalytic component of MPF (p34cdc2) is present within microtubule-rich portions of hemipteran ovarioles, we have found that neither this protein nor its regulatory partner (cyclin B) co-purify with microtubules during taxol-mediated microtubule isolation.

Distribution and subcellular localization of high-molecular-weight microtubule-associated protein-2 expressing exon 8 in brain and spinal cord.

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 ultrastructural 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.

Abnormal phosphorylation of tau and the mechanism of Alzheimer neurofibrillary degeneration: sequestration of microtubule-associated proteins 1 and 2 and the disassembly of microtubules by the abnormal tau.

The microtubule-associated protein (MAP) tau is abnormally hyperphosphorylated in Alzheimer disease and accumulates in neurons undergoing neurofibrillary degeneration. In the present study, the associations of the Alzheimer-hyperphosphorylated tau (AD P-tau) with the high molecular weight MAPs (HMW-MAPs) MAP1 and MAP2 were investigated. The AD P-tau was found to aggregate with MAP1 and MAP2 in solution. The association of AD P-tau to the MAPs resulted in inhibition of MAP-promoted microtubule assembly. However, unlike the coaggregation of AD P-tau and normal tau, the association between AD P-tau and the HMW-MAPs did not result in the formation of filaments/tangles. The affinity of the tau-AD P-tau association was higher than that of HMW-MAPs-AD P-tau because normal tau inhibited the latter binding. The association between AD P-tau and the HMW-MAPs also appeared to occur in situ because these proteins cosedimented from the Alzheimer brain extracts, and, in the sediment, the levels of the HMW-MAPs correlated with the levels of AD P-tau. These studies suggested that the abnormally phosphorylated tau can sequester both normal tau and HMW-MAPs and disassemble microtubules but, under physiological conditions, can form tangles of filaments only from tau.

High Molecular Weight Microtubule-associated Proteins Contain O-Linked N-Acetylglucosamine

We have examined the post-translational modification of high molecular weight microtubule-associated proteins (MAPs) have shown that MAP1, MAP2, and MAP4 are glycosylated. The presence of carbohydrate residues on these proteins was indicated by labeling with biotin hydrazide following periodate oxidation, a specific and well established method for detecting saccharide moieties on proteins. Both MAP2 and MAP4 were also labeled in vitro by UDP-[3H]galactose in the presence of galactosyltransferase. Labeling by galactosyltransferase indicated that MAP2 and MAP4 contained terminal nonreducing GlcNAc residues, and they appeared to be O-linked to the proteins as shown by their sensitivity to beta-elimination. Chromatographic analysis showed that the GlcNAc residues were directly linked to the proteins as monosaccharides. Thus, we have added MAP2 and MAP4 to the list of intracellular O-GlcNAc-modified proteins, which includes other cytoskeletal proteins such as cytokeratins 8, 13, and 18 and neurofilament proteins NF-L and NF-M. We further characterized the O-GlcNAc modification of MAP2, and stoichiometric analysis indicated that nearly 10% of the MAP2 isolated from rat brain is modified by O-GlcNAc. However, this estimate is thought to reflect the minimal level of O-GlcNAc modification present on MAP2. We have also shown that both the O-GlcNAc and biotin hydrazide-reactive carbohydrate moieties are located on the projection domain of MAP2. Three O-GlcNAc-containing peaks were observed following fast protein liquid chromatography of a tryptic digest of MAP2, suggesting that multiple modification sites exist. The specific modification sites and functional significance of the O-GlcNAc glycosylation on the high Mr MAPs remain to be determined.

Genomic structure of human microtubule-associated protein 2 (MAP-2) and characterization of additional MAP-2 isoforms.

We have determined that the gene for human microtubule-associated protein 2 (MAP-2) spans 19 exons, including 6 exons identified in this study, 1-4, 8, and 13; all six of these exons are transcribed. The alternative splicing of coding exons generates a greater diversity of MAP-2 transcripts and isoforms. The first three exons encode alternate 5' untranslated regions that can be spliced to additional untranslated sequences contained in exons 4 and 5. Exons 8 and 13 are transcribed in human fetal spinal cord, adult brain, MSN cells, and rat brain, and each exon maintains an open reading frame with both high and low molecular weight MAP-2 isoforms. Antibodies generated to synthetic peptides of exons 8 and 13 demonstrate that these exons are translated and MAP-2 isoforms containing these exons are generated.

The process of reinnervation in the dentate gyrus of adult rats: gene expression by neurons during the period of lesion-induced growth.

Neurons in the hippocampal dentate gyrus are extensively reinnervated following the destruction of their normal inputs from the ipsilateral entorhinal cortex (EC). The present study evaluates gene expression by dentate granule neurons and the neurons giving rise to the sprouting connections during the period of synapse growth. Adult male rats were prepared for in situ hybridization at 2, 4, 6, 8, 10, 12, 14, 20, and 30 days following unilateral EC lesions. Sections were hybridized using 35S-labeled cRNA probes for mRNAs that encode proteins thought to be important for neuronal structure and/or synapse function, including (1) mRNAs that are normally present in dendrites--the mRNAs for the high molecular weight microtubule-associated protein 2 (MAP2) and the alpha-subunit of calcium/calmodulin-dependent protein kinase II (CAMII kinase), (2) mRNAs that are upregulated in neurons that are regenerating their axons (T alpha 1 tubulin and F1/GAP43) and (3) mRNAs for proteins that are the principal constituents of neurofilaments and microtubules (the low molecular weight neurofilament protein NF68 and beta-tubulin). Although there were small changes in the levels of labeling for the mRNAs that are normally present in dendrites, there were no dramatic increases in the levels of any of the mRNAs either in dentate granule cells or in neurons giving rise to the reinnervating fibers at any postlesion interval. These results indicate that neurons in mature animals can substantially remodel their synaptic terminals and their dendrites in the absence of large-scale changes in gene expression (at least as measured by steady-state mRNA levels at various time points).

Human microtubule-associated protein-2c localizes to dendrites and axons in fetal spinal motor neurons.

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.

Two novel HMW MAP2 variants with four microtubule-binding repeats and different projection domains

The brain microtubule-associated protein MAP2 is composed of two high molecular (MAP2a and b) and one low molecular (MAP2c) weight isoforms. All these forms were thought to contain three repeated microtubule-binding domains in their C-terminal region but a MAP2c variant containing four repeats has recently been identified. We report here the existence of two high molecular weight MAP2 isoforms with four microtubule-binding domains in the sensory neuronal cell line ND 7/23. A stretch of 135 bp is missing in one of these forms suggesting that several HMW MAP2 variants can be produced by alternative splicing.

Dendrites as compartments for macromolecular synthesis.

Dendrites as compartments for macromolecular synthesis.

Structure, regional and developmental expression of rat MAP2d, a MAP2 splice variant encoding four microtubule-binding domains

MAP2, a major component of microtubule polymers in neurons consists of high molecular weight (HMW) proteins MAP2a, MAP2b and a low molecular weight (LMW) MAP2c, expressed in the developing brain. These isoforms are produced from a single gene by alternative splicing and share identical C-termini encompassing 3 tandem repeats, critical in microtubule binding. We describe the structure, regional and developmental expression of a novel MAP2 splice variant, MAP2d, containing an insertion whose sequence is homologous to the three and four repeats of MAP2 and Tau respectively. This insertion is absent from the mRNAs encoding HMW MAP2. MAP2d mRNAs are expressed at higher levels than MAP2c in all adult nervous tissues of the rat, and are found at low levels in glial cell cultures when compared to primary cultures of cerebellar neurons. Splicing of the fourth repeat in mature Tau precedes that in MAP2d during rat brain development. The tardive expression of a four microtubule-binding domain LMW MAP2 suggests it could play in extended neurites a similar role as mature Tau in axons.