Microtubule-associated Protein 1B (MAP1B) Is Required for Dendritic Spine Development and Synaptic Maturation

Background: Microtubule-associated protein 1B (MAP1B) is a protein that is prominently expressed during early neuronal development but in adult brain remains in areas with high synaptic plasticity.

Results: MAP1B plays an important role in dendritic spine formation and synaptic maturation.

Conclusion: A novel function for MAP1B in regulating dendritic spine morphology and synaptic function is indicated.

Significance: MAP1B could contribute to adult brain plasticity.

Abstract

Microtubule-associated protein 1B (MAP1B) is prominently expressed during early stages of neuronal development, and it has been implicated in axonal growth and guidance. MAP1B expression is also found in the adult brain in areas of significant synaptic plasticity. Here, we demonstrate that MAP1B is present in dendritic spines, and we describe a decrease in the density of mature dendritic spines in neurons of MAP1B-deficient mice that was accompanied by an increase in the number of immature filopodia-like protrusions. Although these neurons exhibited normal passive membrane properties and action potential firing, AMPA receptor-mediated synaptic currents were significantly diminished. Moreover, we observed a significant decrease in Rac1 activity and an increase in RhoA activity in the post-synaptic densities of adult MAP1B^+/− mice when compared with wild type controls. MAP1B^+/− fractions also exhibited a decrease in phosphorylated cofilin. Taken together, these results indicate a new and important role for MAP1B in the formation and maturation of dendritic spines, possibly through the regulation of the actin cytoskeleton. This activity of MAP1B could contribute to the regulation of synaptic activity and plasticity in the adult brain.

Introduction

Microtubules (MTs)⁴ are non-covalent cytoskeletal polymers composed of α- and β-tubulin heterodimer subunits that are assembled into linear protofilaments. These are essential structures for the maintenance of neuronal morphology and in developing neurons. MTs are highly dynamic structures that are involved in neurite extension and the establishment of neuronal polarity (1). By contrast, in differentiated and mature neurons, axonal and dendritic MTs become more stable due to their interactions with structural MT-associated proteins (MAPs) (2) such as Tau and MAP2, respectively (3). Thus, MAPs contribute to neuronal morphology by regulating the balance between MT stability and plasticity in neuronal processes (4). These proteins are differentially expressed during brain development, displaying a transition from juvenile to adult isoforms that correlates with the maturation of the central nervous system (CNS) and the stabilization of neuronal circuitry (5, 6).

MAP1B (7) is the first MAP to be expressed strongly in the nervous system during embryonic development (8). Subsequently, its expression is developmentally down-regulated until it almost disappears from the axon during the formation of synaptic contacts between neurons in the CNS (9–13). The role of MAP1B in axonogenesis has been widely studied (for review, see Ref. 14). Thus, suppression of MAP1B with antisense oligonucleotides inhibits laminin enhanced axon growth (15), and there is a significant delay in axon outgrowth and a reduced rate of axon elongation in cultured hippocampal pyramidal neurons from MAP1B-deficient mice (16, 17).

MAP1B was recently shown to regulate Rac1 activity during axonal outgrowth through its interaction with Tiam1, a Rac1-GEF (guanosine nucleotide exchange factor). Accordingly, MAP1B deficiency results in a decrease in the activity of the Rho-GTPases Rac1/cdc42 and an increase in RhoA activity (18). These results are consistent with members of the Rho family of small GTPases, including RhoA, Rac1, and cdc42, regulating the cross-talk between actin and MTs in developing neurons, affecting axon specification, guidance, and elongation (19, 20).

MAP1B has been described in the post-synaptic compartment (21–23), a scaffolding specialization of the neuronal synapses at the tip of the dendritic spine. Dendritic spines are actin-rich membrane protrusions that extend from the dendritic shaft with a globular head and thin neck. As a basic functional unit of the excitatory synapse, dendritic spines are critical for most excitatory nerve transmission in the brain (24). Spines are dynamic structures that undergo actin-dependent changes in shape, size, and number, and thus, they represent major sites of structural synaptic plasticity (25). Rho-GTPases such as Rac1 and RhoA are key players in regulating the dynamics of the actin cytoskeleton in neuronal spine morphogenesis (26, 27). Moreover, Tiam1 has also been implicated in NMDA receptor activity-dependent structural plasticity (28).

Although it is well established that F-actin is the major cytoskeletal component of dendritic spines (29), it was previously thought that MTs were absent from these structures and that they were confined to the dendritic shaft in mature neurons (30, 31). However, the transient presence of dynamic MTs in dendritic spines of adult neurons was recently confirmed, implicating MTs in spine formation and plasticity (32–34). The presence of a MT-binding protein in dendritic spines was also demonstrated recently, participating in spine morphogenesis (34).

Although the role of MAP1B in axonal development has been studied extensively, little is known of its function in postsynaptic compartments and particularly in dendritic spines. Based on the activity of MAP1B as a scaffold protein (35–37), its interaction with actin filaments (36, 38), and the recently described role of MTs in dendritic spine development, we postulated that MAP1B may be involved in regulating the morphogenesis and plasticity of postsynaptic elements. As such, we demonstrate here that MAP1B plays an important role in dendritic spine formation and synaptic maturation by regulating of actin cytoskeleton.

EXPERIMENTAL PROCEDURES

Primary Antibodies

The following primary antibodies (Ab) were used here: mouse anti-α-tubulin Ab (1:2000; Sigma); mouse Ab anti-α-actin (Sigma); goat Ab against MAP1B (N-19, 1:2000; Santa Cruz Biotechnology); mouse anti-HMW-MAP2 (clone HM-2, 1:5000; Sigma); goat anti-Tau (C-17, 1:1000; Santa Cruz Biotechnology); mouse Ab against phosphorylated neurofilament heavy subunit (SMI-34, 1:1000; Sternberger Monoclonals); rabbit anti-GEF-H1 (1:1000; Abcam); rabbit anti-Tiam1 (clone C-16, 1:200; Santa Cruz Biotechnology); anti-cofilin mAb (1:1000; a generous gift of Dr. James Bamburg, Colorado State University); rabbit anti-phospho-cofilin (Ser-3, clone 77G2, 1:1000; Cell Signaling); antibodies against Rac1 and RhoA (1:500; Cytoskeleton, Inc.).

The phosphorylation-independent Tau monoclonal antibody 7.51 (39) was kindly provided by Dr. C. M. Wischik. The monoclonal antibodies Tau-1 and Tau-5 were purchased from Chemicon International and Calbiochem, respectively.

Cell Culture

Cultures of dissociated hippocampal pyramidal cells from wild type (WT) and MAP1B knock-out (KO) mouse embryonic brains were prepared as previously described (40). The hippocampus was removed and digested with papain and DNase (Worthington Biochemical Corp.). 50,000 cells/cm² were plated onto 12-mm glass coverslips (precoated with 100 μg/ml poly-l-lysine) in wells containing Neurobasal medium with 10% horse serum. After 3 h the plating medium was replaced with Neurobasal medium supplemented with 2 mm l-glutamine, 2 mm d-pyruvate, 1% N2-supplement and 2% B27-supplement (GIBCO), 100 units/ml penicillin, and 100 mg/ml streptomycin. Hippocampal cells were cultured over a monolayer of astrocytes prepared from the cortex of newborn (P0) Swiss-Webster mice. The cortices were treated with trypsin and DNase, and the tissue pieces were dissociated mechanically and passed through a 70-μm mesh filter (BD Falcon) in minimum Eagle's medium supplemented with 10% horse serum, 0.6% glucose, 0.2 mm glutamine, and antibiotics. 400,000 cells/well were seeded in 6-well plates and grown for 21 days until a confluent monolayer of astrocytes was established. All culture media and supplements were purchased from Invitrogen. Neuronal cultures were maintained in a humidified 37 °C incubator with 5% CO₂ until 21 days in vitro (DIV).

Immunofluorescence

At 21 DIV, primary cells were fixed with 4% paraformaldehyde, 4% sucrose for 20 min at 4 °C. The cells were incubated with phosphate-buffered saline (PBS), 0.1% Triton X-100 for 5 min and then blocked with PBS, 5% (w/v) bovine serum albumin (BSA) for 1 h. Subsequently, the cells were incubated with primary antibodies raised against the proteins indicated and diluted in PBS, 1% BSA. The fluorescent secondary antibodies (Molecular Probes, Invitrogen) were used at a dilution 1:400. Phalloidin-TRITC (Sigma) and phalloidin-FITC (Molecular Probes) were both used at a concentration of 1:200 to label the actin cytoskeleton. Cells were analyzed on a Zeiss LSM 510 META confocal scanning microscope.

Protein Extracts and Western Blotting

Protein extracts were prepared from the hippocampus, corpus callosum (white matter), or cortex (gray matter) of adult mice in 20 mm HEPES (pH 7.4) containing 0.1 mm NaCl, 10 mm NaF, 1 mm Na₃VO₄, 5 mm EDTA, 1 mm okadaic acid, and protease inhibitors (2 mm phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mg/ml pepstatin). Protein extracts were boiled for 5 min in electrophoresis sample buffer (50 mm Tris-HCl (pH 6.8), 100 mm dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol) and then separated by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman). The membranes were blocked with 5% dried nonfat milk in PBS and 0.1% Tween 20 and probed for 1 h at room temperature with the primary antibody in the blocking solution. After three washes, the membrane was incubated with a horseradish peroxidase secondary antibody (DAKO) followed by several washes in PBS-Tween 20. Antibody binding was visualized by ECL (PerkinElmer Life Sciences) and quantified by densitometry.

Image Analysis and Dendritic Spine Quantification

To analyze the number, length, and head and neck morphology of dendritic spines, we acquired confocal images of wt and knock-out neurons at 21 DIV using an oil immersion 63× objective. Photos were taken at a resolution of 1024 × 1024 pixels and a pinhole of 0.60 μm.

To perform three-dimensional reconstructions of spine morphology, Z-stacks of 13 planes were generated, each separated by 0.150 μm. Images were subsequently deconvoluted using the Huygens program and reconstructed using the IDL time Calc program. The classification of dendritic spine type was carried out according to the following criteria; 1) mushroom spines were those with a large head of a diameter greater than 0.75 μm and with a short narrow neck, 2) stubby spines were those with no obvious constriction between the head and the shaft, 3) thin spines were those with a small head of a diameter between 0.5–0.7 μm and a long narrow neck, and 4) filopodia were defined as thin protrusions longer than 1.5 μm and with no distinct head. Spine density and length were quantified in 3 dendritic branches per neuron (at least 3 independent experiments, 10 neurons per experiment).

Immunoprecipitation

The hippocampus of an adult mouse brain was homogenized in 0.7 ml of cold immunoprecipitation buffer (20 mm Tris (pH 7.5), 0.5% Triton X-100, 100 mm NaCl), and the homogenate was then centrifuged for 15 min at 13,500 rpm and 4 °C. The supernatant obtained was considered as the total cell lysate.

To 400 μg of the supernatant, 1 μg of an antibody against MAP1B or a mixture of Tau antibodies (Tau-1, Tau 7.51, Tau-5) was added in a final volume of 1 ml. As a negative control, goat or mouse IgG isotype control antibody (for MAP1B or Tau immunoprecipitation experiments, respectively; Santa Cruz Biotechnology) was incubated with the extract. The solution was vortexed and incubated for another 1 h at 4 °C, and 20 μl of 50% Protein A-agarose bead solution was then added, mixed, and incubated with agitation for 30 min at 4 °C. The beads were recovered by centrifugation for 15 min at 13,500 rpm and 4 °C, and the supernatant was removed. The pellet was washed twice with the immunoprecipitation buffer and resuspended in 30 μl of 2× concentrated electrophoresis sample buffer. The proteins were separated by gel electrophoresis, and the fractionated proteins then characterized in Western blots.

Synaptosome Purification

The protocol used to purify synaptosome fractions from adult rat brain is based on well established methods that have been described previously (41, 42). The whole adult mouse brain was homogenized at 800 rpm/7 strokes in a Dounce glass homogenizer in 4 vol/g of buffer A (0.32 mm sucrose, 1 mm MgCl₂, 0.5 mm CaCl₂, 1 mm NaHCO₃, 1 mm dithiothreitol, and protease inhibitors (2 mm phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mg/ml pepstatin). After the addition of 10 vol/g of buffer A, the homogenate was centrifuged for 10 min at 1400 × g to recover the supernatant S1 and the pellet P1. P1 was resuspended in 4 vol/g of buffer A, homogenized at 800 rpm/3 strokes, and recentrifuged for 10 min at 700 × g. The resulting supernatant was combined with S1 and centrifuged for 10 min at 13,800 × g. The resulting supernatant (S2) was separated from the pellet P2 and centrifuged at 100,000 × g for 1 h. The supernatant obtained (S3) constitutes the cytosolic fraction. P3 was resuspended in 24 ml/10 g wet weight of buffer B (0.32 mm sucrose, 1 mm NaHCO₃, 1 mm EGTA, 1 mm dithiothreitol, and protease inhibitors (2 mm phenylmethylsulfonyl-fluoride, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mg/ml pepstatin)) and homogenized to obtain the crude synaptosome fraction. To separate the synaptic components, the synaptosome fraction was diluted 10× in ice-cold 0.1 mm CaCl₂. The same volume of a 2× concentrated solution (40 mm Tris (pH 6.0) and 2% Triton X-100) was added. This was incubated for 30 min on ice with mild agitation and centrifuged for 30 min at 30,000 rpm in a TL856 rotor. The pellet constituted the synaptic junctions, and the supernatant contained the extrasynaptic proteins. The pellet was washed with 20 mm Tris (pH 6.0) and 1% Triton X-100 and resuspended in 500 μl of 20 mm Tris (pH 8.0) and 1% Triton X-100. This solution was then incubated on ice for 30 min with mild agitation and centrifuged at 30,000 rpm in a TL100.1 rotor. The resulting pellet was resuspended in 50 μl of lysis buffer (50 mm Tris, 150 mm NaCl, 1% Triton X-100, 0.5% deoxycholate (pH 7.2)), and this last fraction (the postsynaptic density fraction) was analyzed in Rho-GTPase activity assays and Western blots. Synaptophysin was used to exclude the presence of presynaptic matrix and postsynaptic density fraction 95 as a marker of the postsynaptic density fraction.

Assays of Rho-GTPase Activity

The Rac1 activity assay was performed as described previously (43). Briefly, the synaptosome fraction from adult mice (2 months of age) was lysed at 4 °C in 50 mm Tris, 150 mm NaCl, 1% Triton X-100, 0.5% deoxycholate (pH 7.2). GTP-bound Rac1 was affinity-purified from cell lysates at 4 °C using an immobilized GST fusion of the Rac1-binding domain of murine p21-activated protein kinase (PAK), which binds Rac1-GTP but not Rac1-GDP. Bound proteins were separated by 12% SDS-PAGE and immunoblotted with anti-Rac1 antibodies (Rac1 activation assay Biochem kit, Cytoskeleton, Inc). Pulldown assays and immunoblots for activated RhoA were performed as described for Rac1 but using an immobilized GST fusion of the Rho binding domain of the Rho effector protein, rhotekin. The Rho binding domain motif binds specifically to the GTP-bound form of RhoA. Total RhoA and RhoA-GTP were detected with mouse anti-RhoA antibodies (RhoA activation assay Biochem kit, Cytoskeleton, Inc.).

Recordings of Miniature Excitatory Postsynaptic Currents

Voltage-clamp whole-cell recordings were obtained from 21 DIV hippocampal neurons from MAP1B^+/+ and MAP1B^−/− mice at −60 mV in the presence of 100 μm 2-amino-5-phosphonovaleric acid; Sigma) and tetrodotoxin (1 μm, Sigma). Spontaneous events were acquired and analyzed with pClamp software (Molecular Devices).

Solutions

The recording chamber was perfused with aCSF: 119 mm NaCl, 2.5 mm KCl, 2.5 mm CaCl₂, 1.3 mm MgCl₂, 26 mm NaHCO₃, 1 mm NaH₂PO₄, 11 mm glucose, 0.1 mm picrotoxin (pH 7.4), gassed with 5% CO₂, 5% O₂. Patch recording pipettes (3–6 megaohms) were filled with 115 mm cesium methanesulfonate, 20 mm CsCl, 10 mm HEPES, 2.5 mm MgCl₂, 4 mm Na₂ATP, 0.4 mm Na₃GTP, 10 mm sodium phosphocreatine, 0.6 mm EGTA (pH 7.25).

Nocodazole Treatment

Hippocampal neurons obtained from wild type mice were cultured for 2 weeks and then treated with nocodazole (Sigma) added for 3 h at a final concentration of 30 μg/ml. NIE-115 mouse neuroblastoma cells (ATCC) were cultured in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 °C in 5% CO₂. Cell differentiation was induced by overnight serum deprivation, and NIE-115 cells were treated with nocodazole (10 μg/ml) for 20 min. Cell extracts were prepared in cold immunoprecipitation buffer and centrifuged for 15 min at 13,500 rpm and 4 °C. The supernatant was considered as the total cell lysate. The immunoprecipitation was performed as described previously.

Statistical Analysis

All the statistical tests were performed, and all the graphs were constructed with GraphPad Prism5 software. Statistical analysis was performed using parametric (Student's t test) and non-parametric (Mann-Whitney test) tests depending on normality of the data sets (assessed with the Shapiro-Wilk test) at a significance level of p ≤ 0.05. p values are indicated in the graphs, and p < 0.05 is considered indicative of a statistically significant difference. All data are expressed as the means ± S.E.

RESULTS

MAP1B Is Present in Dendritic Compartments of Mature Neurons

The presence and distribution of MAP1B in adult brain was analyzed by immunofluorescence in brain slices from wild type mice. The nature of the cellular extensions was determined by staining with antibodies against dendritic and axonal marker proteins, HMW-MAP2 and phosphorylated neurofilament heavy subunit, respectively. MAP1B was detected primarily in the dendrites of hippocampal neurons (Fig. 1, A–H). To confirm these results, we isolated white matter and gray matter from adult wild type mouse brain and analyzed the presence of MAP1B in these regions. Although gray matter is enriched in the somatodendritic compartment, it also contains fractions of neuropil containing unmyelinated and myelinated axons and glial cells as well as capillaries. Using neurofilament heavy subunits and HMW-MAP2 as axonal and dendritic markers, we demonstrated that the gray matter fraction did not contain high levels of axonal material, but rather, it was enriched in somatodendritic compartments. In addition, MAP1B is mainly expressed in neurons and only weakly in glial cells, whereas it is absent from capillaries (44–47). Thus, MAP1B was concentrated in the gray matter, and it was less prevalent in the white matter, as reported previously (48, 49).

FIGURE 1.

MAP1B is present in adult brain. A–H, shown are representative immunofluorescence images of WT mouse brain slices (30 μm thick) from 2-month-old animals. The slices were stained with antibodies against the specific dendritic marker HMW-MAP2 (green, A) and specific axonal marker neurofilaments (NF) (green, E) and against MAP1B (red, B and F) and βIII-tubulin (blue, C and G). MAP1B is located in the apical dendrites of CA1 hippocampal neurons (D–H). Scale bar = 20 μm. I, a Western blot demonstrates the presence of MAP1B in both white and gray matter in the adult mouse, although staining is more abundant in the gray matter (upper panel). We used HMW-MAP2 as a dendritic marker (bottom panel) and phosphorylated neurofilament heavy subunit as an axonal marker in the white matter.

MAP1B Is Present in Some but Not All Dendritic Spines

Dynamic MTs and MT-binding proteins have been recently detected in dendritic spines of mature neurons (32–34). Hence, we examined the detailed subcellular distribution of MAP1B in mature dendrites. Immunostaining of 21 DIV hippocampal neurons with antibodies directed against MAP1B (N19) and HMW-MAP2 (as a dendritic marker) revealed that MAP1B predominantly localized within the dendritic compartment (Fig. 2, A–D). However, immunostaining using anti-MAP1B (N19) and phalloidin (which targets F-actin and was used as a marker of dendritic spines and filopodia) demonstrated that MAP1B was occasionally observed in a small fraction of the dendritic protrusions (arrowhead in Fig. 2, E–G), colocalizing with F-actin (Fig. 2G). These F-actin-positive structures fulfilled the morphological and morphometric criteria to be considered dendritic spines. Immunostaining of MAP1B knock-out neurons was performed as a negative control (Fig. 2, H–J). The number of dendritic spines that are positive for MAP1B staining represents 1%. Interestingly, in the pioneering studies demonstrating the presence of MTs in dendritic spines, the percentage of invasion of MTs in dendritic spines on fixed cells was estimated to be 1–4% (32–34, 50).

FIGURE 2.

MAP1B is detected in a small percentage of dendritic spines. A–D, confocal images are shown of 21 DIV hippocampal WT neurons stained with antibodies against the specific dendritic marker HMW-MAP2 (green, A), MAP1B (red, B), and βIII-tubulin (blue, C). HMW-MAP2 labeling reveals the presence of MAP1B in dendrites (D). Scale bar = 20 μm. E–J, confocal images of 21 DIV hippocampal neurons stained with phalloidin (green, E) and MAP1B (red, F) are shown. MAP1B is present in some dendritic spines (arrows), co-localizing with phalloidin in the spine head (G). MAP1B immunostaining of MAP1B knock-out neurons was performed as a negative control (H–J). Scale bar = 10 μm.

MAP1B Is Important for Proper Spine Formation

After the detection of MAP1B in some dendritic spines, we investigated whether decreased expression of MAP1B could affect dendritic spine morphogenesis. We previously characterized MAP1B-deficient mice generated by gene trapping (51). Homozygous mice die perinatally, whereas heterozygous mice develop with no evident neurological problems. We, therefore, established long term cultures of hippocampal neurons derived from MAP1B homozygous and wild type littermates in the presence of an astrocyte monolayer. Using this culture model we analyzed the morphology and number of dendritic protrusions in the knock-out and wild type neurons. When hippocampal WT and MAP1B^−/− neurons were stained at 21 DIV with HMW-MAP2 (dendritic marker) and phalloidin (dendritic protrusion marker; Fig. 3, A–D), there were significantly fewer dendritic protrusions on neurons from MAP1B^−/− mice than on those from MAP1B^+/+ mice (Fig. 3E). Moreover, the filopodia-like protrusions of knock-out neurons were generally longer (>2 μm) than those of the wild type controls (Fig. 3F). Three-dimensional reconstructions were performed to classify the different dendritic protrusions observed. Protrusions from control neurons predominantly exhibited the morphological characteristics of mature mushroom-shaped and stubby spines, with a large head and no distinguishable neck (Fig. 4, A, C, and E). In contrast, most protrusions from MAP1B^−/− neurons corresponded to immature spines and were classified as filopodia, long thin protrusions without a distinguishable head (Fig. 4, B, D, and E). Taken together, these observations are consistent with the spine length measurements described above.

FIGURE 3.

MAP1B is important for proper spine formation. A and B, confocal microscopy images show the morphology of 21 DIV hippocampal MAP1B^+/+ (A) and MAP1B^−/− neurons (B) stained with phalloidin (green) and HMW-MAP2 (red). Scale bar = 20 μm. C and D, a magnified image is shown; note that WT neurons have mature spine-like protrusions with a defined head (C), whereas KO neurons exhibit long and thin filopodia-like protrusions (D). Scale bar = 2 μm. E, quantitative analyses indicate that MAP1B-deficient neurons exhibit a lower density of dendritic protrusions (*, p ≤ 0.001, Student's t test). F, quantitative analyses demonstrate that the protrusions in MAP1B^−/− neurons are longer than in control neurons (n = 10 neurons from three independent experiments per condition).

FIGURE 4.

Three-dimensional reconstruction of dendritic spines in 21 DIV hippocampal neurons. A–D, deconvolved confocal images are shown of MAP1B^+/+ (A) and MAP1B^−/− neurons (B), created using the Huygens program and reconstructed with the IDL time Calc program. Scale bar = 5 μm. Note that dendrites from wild type neurons have different types of spines, including mushroom, stubby, and branched, constituting the population of mature spines (arrows in C). By contrast, knock-out neurons predominantly generate long protrusions without a distinguishable head (arrows in D). Scale bar = 2 μm. E, a graph shows the percentage of the different types of spines found in dendrites of MAP1B^+/+ and MAP1B^−/− neurons (n = 10 neurons from three independent experiments per condition).

The Absence of MAP1B Decreases Postsynaptic Function

Electrophysiological studies were performed to determine whether the decrease in mature dendritic spine number in the MAP1B knock-out mice affects synaptic function. We first examined the passive membrane properties of MAP1B^−/− and control neurons. Whole-cell recordings using a current-clamp configuration revealed no significant alterations in membrane capacitance (related to cell membrane surface), input resistance, or resting membrane potential (related to resting ionic conductances) in MAP1B^−/− hippocampal neurons (supplemental Fig. 1, A–C). Moreover, we found no differences in the action potential threshold, amplitude, or shape between control and MAP1B^−/− neurons (supplemental Fig. 2, A–C).

We also analyzed the postsynaptic properties of control and MAP1B^−/− neurons (Fig. 5A), recording miniature synaptic currents mediated by AMPA receptors under voltage-clamp at −60 mV in the presence of a selective NMDA receptor antagonist (2-amino-5-phosphonovaleric acid) and tetrodotoxin (to prevent action potential firing). The amplitude of miniature excitatory postsynaptic currents (mEPSC) recorded in knock-out neurons was significantly smaller than that of control neurons (Fig. 5), an effect that was observed throughout the whole distribution of mEPSC amplitudes (see the left-shift in the cumulative distribution; Fig. 5B). This effect suggests a decrease in the number of functional AMPA receptors at synapses in knock-out neurons, in line with the relative predominance of immature dendritic spines in these neurons.

FIGURE 5.

Recordings of miniature synaptic currents from wild type and MAP1B^−/− hippocampal neurons. Spontaneous events were recorded in 21 DIV neurons from MAP1B^+/+ and MAP1B^−/− mice in patch-clamped conditions at −60 mV in the presence of 2-amino-5-phosphonovaleric acid (100 μm) and tetrodotoxin (1 μm). A, representative traces from ∼0.1 s of recordings from 21 DIV neurons from MAP1B^+/+ and MAP1B^−/− mice are shown. B, cumulative distribution of mEPSC amplitude recorded from wild type (n = 10374 minis from 22 cells) and MAP1B^−/− (n = 5304 minis from 25 cells) hippocampal neurons (*, p ≤ 0.0001, Kolmogorov-Smirnov test) are shown. C, average mEPSC amplitude recorded from MAP1B^+/+ (22 neurons) and MAP1B^+/− (25 neurons) hippocampal neurons are shown (*, p ≤ 0.01, Mann-Whitney test).

MAP1B-deficient Mice Exhibit Weaker Postsynaptic Rac1 Activity and Stronger RhoA Activity

Given the observed morphological and morphometric abnormalities in dendritic spines of MAP1B-deficient neurons, we investigated the function of RhoGTPases in these neurons. Active Rac1 and RhoA were measured in the postsynaptic density fraction of adult WT and MAP1B^+/− animals. We previously described decreases in Rac1 and Cdc42 activity and increased RhoA activity in MAP1B-deficient hippocampal neurons, resulting in impaired axonal elongation in short term cell cultures (18). Moreover, several previous studies have shown Rho-GTPases to be important regulators of the actin cytoskeleton that contributes to the structural plasticity of excitatory synapses (26, 27). Specifically, Rac1 and RhoA are key molecules in the regulation and dynamics of dendritic spines (26, 52–54).

Levels of MAP1B were first measured in Western blots of extracts from wild type and heterozygous MAP1B^+/− mice. Knock-out animal could not be used for these experiments as the MAP1B^−/− mouse suffers early postnatal lethality. As expected, there was less MAP1B in the MAP1B^+/− extracts than in the wild type extracts (Fig. 6A).

FIGURE 6.

MAP1B-deficient mice exhibit weaker synaptosome Rac1 activity and stronger RhoA activity. A, a Western blot demonstrates higher MAP1B levels in the adult hippocampus of WT (^+/+) mice (upper panel) than in MAP1B^+/− animals. α-Tubulin was used as loading control. Quantitative analysis confirmed a significant decrease in MAP1B in heterozygous MAP1B^+/− extracts (*, p ≤ 0.01, Student's t-test). B, a Western blot shows lower Rac1-GTP levels in synaptosome extracts from the adult brain of heterozygous MAP1B^+/− animals versus those of the WT (^+/+) mice. Total Rac1 was used as a loading control. Quantitative analysis confirmed the significant decrease in Rac1 activity in heterozygous MAP1B^+/− extracts (*, p ≤ 0.05, Student's t test). C, shown is increased RhoA activity in synaptosome fractions from MAP1B^+/− versus control samples. Total RhoA was used as a loading control. Quantitative analysis indicated a significant increase in RhoA activity in heterozygous (^+/−) fractions (*, p ≤ 0.05, Student's t test). D, shown are total cofilin and phosphocofilin levels in synaptosome extracts obtained from the adult brain of heterozygous MAP1B^+/− and WT (^+/+) mice, as detected by Western blot. Phosphocofilin levels decreased in heterozygous MAP1B animals when compared with WT controls (right panel; *, p ≤ 0.05, Mann-Whitney test). Cofilin was used as a loading control. n = 4 animals per genotype in A–D.

To measure Rac1 activity, we performed a pulldown assay using a GST fusion protein corresponding to the p21 binding domain (residues 67–150) of murine PAK1, a Rac effector that binds to Rac1-GTP with a high affinity but not to Rac1-GDP. Rac1 activity was significantly lower in postsynaptic density fraction extracts from MAP1B^+/− mice than in WT extracts, whereas the total Rac1 protein was similar in both (Fig. 6B). Quantitative analyses revealed a 40% decrease in Rac1 activity in MAP1B^+/− versus control samples (Fig. 6B, right panel). By contrast, RhoA activity was stronger in MAP1B^+/− than WT extracts, and a 45% increase in RhoA activation in MAP1B^+/− extracts over the activity in the WT sample was quantified (Fig. 6C, right panel). This increase was not due to enhanced RhoA expression, as the overall levels of RhoA were comparable in both WT and MAP1B^+/− mice (Fig. 6C).

We also investigated whether the activity of downstream effectors of Rac1 signaling, such as cofilin, was altered in MAP1B^+/− animals. Cofilin protein binds to F-actin and controls the turnover of the subunits in the polymer, increasing depolymerization at the end of the filament and severing long actin filaments. Cofilin is rendered inactive when it is phosphorylated by the protein kinase LIMK-1 (55), increasing the density and modifying the morphology of dendritic spines (56). We quantified the amount of phosphorylated cofilin and total cofilin in the post-synaptic fractions from MAP1B^+/− and MAP1B^+/+ animals. Although no differences in the levels of total cofilin were detected, there was less phospho-cofilin in the MAP1B^+/− extracts (Fig. 6D), consistent with the increase in the number of immature dendritic spines found in MAP1B^−/− neurons.

MAP1B may increase and decrease Rac1 and RhoA activity, respectively, by binding to a GEF that acts on Rac1 or RhoA proteins. Tiam1 is a Rac1 GEF (57) that co-immunoprecipitates with MAP1B in extracts of young mice brains (18). Indeed, Tiam1 and MAP1B were co-immunoprecipitated from hippocampal extracts of adult mice (Fig. 7, A and B).

FIGURE 7.

MAP1B co-immunoprecipitates with the GEFs Tiam1 and GEF-H1 irrespective of the presence or absence of tubulin polymers. A and B, shown is co-immunoprecipitation (IP) of MAP1B with Tiam1, the specific Rac1 GEF, and with GEF-H1, the specific RhoA GEF, in Western blots (WB) (A). Immunoprecipitation of Tau protein was performed in parallel to assess its co-immunoprecipitation with either Tiam1 or GEF-H1 (A). Positive controls show MAP1B and Tau immunoprecipitations after the addition of the respective antibodies (B). C, hippocampal neurons were pretreated with nocodazole (30 μm) for 3 h, and MAP1B was immunoprecipitated, showing that Tiam1 and GEF-H1 could be detected in the immunoprecipitate. D, detection of Tiam1 and GEF-H1 after MAP1B immunoprecipitation in N1E-115 neuroblastoma cells pretreated with 10 μm nocodazole for 20 min is shown. Negative controls, performed in the absence of specific antibodies (see “Experimental Procedures”) are shown in the different panels (control). Co-immunoprecipitation of GEFs with MAP1B occurred irrespective of the presence of tubulin polymers.

To explain how the absence of MAP1B could increase RhoA activity in the adult brain, we analyzed MAP1B binding to GEF-H1, which acts on RhoA. GEF-H1 was recently proposed to form a complex with the RhoA protein and MTs (58). Now we have detected that GEF-H1 co-immunoprecipitates with MAP1B in the adult brain (Fig. 7, A and B).

To determine whether MAP1B simply immunoprecipitates with tubulin-binding proteins such as GEF-H1 or Tiam1 through its interaction with tubulin polymers, we performed further immunoprecipitation studies using Tau protein, another MAP present in mature neurons that localizes in dendritic compartments (59). Significantly, the Tau protein did not co-immunoprecipitate with either Tiam1 or GEF-H1 (Fig. 7, A and B).

Similarly, to rule out the possibility that tubulin polymers mediate the co-immunoprecipitation of GEFs with MAP1B, we repeated the immunoprecipitations using cultured neurons pretreated with the MT depolymerizing agent nocodazole. After nocodazole treatment, Tiam1 and GEF-H1 still co-immunoprecipitated with MAP1B (Fig. 7C). However, mature neurons contain a large population of stable MTs resistant to nocodazole and with half-lives exceeding several hours (60). Thus, we repeated these immunoprecipitation studies in a neuroblastoma cell line, N1E-115, pretreated with nocodazole. This approach resulted in complete depolymerization of MTs by nocodazole, yet MAP1B still co-immunoprecipitated with the GEFs of interest (Fig. 7D). Based on these findings, we propose that MAP1B regulates Rac1 and RhoA activities through interactions with GEFs.

DISCUSSION

MAP1B is predominantly expressed during neuronal development, although strong expression is maintained in adult brain regions that exhibit a high degree of plasticity (8, 61, 62). At the subcellular level, MAP1B is mainly present in dendrites of adult neurons (63) where it may contribute to AMPA receptor trafficking through binding to glutamate receptor interacting protein 1 (GRIP) (64, 65). MAP1B has also been detected in postsynaptic densities (23), and it was demonstrated to be a synaptic protein in a proteomic analysis (21, 22). Here, we demonstrate the presence of MAP1B in the adult hippocampus and in mature neurons in culture. In the brain, MAP1B is more abundant in the gray matter, and it is enriched in somatodendritic compartments. Moreover, confocal microscopy revealed the presence of MAP1B in a small percentage of dendritic spines in long-term neuronal cultures.

Dendritic spines are small protrusions whose main cytoskeletal component is actin. Indeed, MTs were assumed to be absent from dendritic spines until the transient entry of dynamic MTs into dendritic spines was recently described (32–34). MAP1B is an MT-associated protein that is implicated in the maintenance of dynamic MT populations (66, 67), and it interacts directly with actin (37, 38). Thus, it is reasonable to hypothesize that MAP1B can enter into dendritic spines in association with either dynamic MTs or actin microfilaments. The movement of MAP1B into dendritic spines appears to be transient based on the small number of MAP1B-positive spines detected. This observation is in agreement with the percentage of MTs detected in dendritic protrusions (∼1% of dendritic protrusions) (33). Therefore, we propose that MAP1B gains access to dendritic spines by accompanying dynamic MTs. Interestingly, a large number of spines have previously found to contain MTs, although the duration for which the MTs remained in each spine is limited to a few minutes (33). This could explain why, although we only detect MAP1B in a small percentage of dendritic spines at a fixed time, the effect seems to be global on all spines. A possible role for dynamic MTs in dendritic spines is supported by the decrease in mature dendritic spines seen in neurons lacking the EB3 protein (32, 34). Moreover, low concentrations of nocodazole suppress the synaptic potentiation induced in response to high frequency stimulation (34). These effects may involve the local regulation of MT dynamics. Interestingly, MAP1B increases the amount of tyrosinated (i.e. more dynamic) MTs through a mechanism involving the interaction of MAP1B with the enzyme tubulin tyrosine ligase (67). It has been proposed that the transient entry of MTs into dendritic spines serves as a signal to locally reorganize the actin cytoskeleton and regulate spine size (33, 34). The postsynaptic accumulation of MAP1B suggests a role in the activity of the synapse, spine formation, and synapse establishment. In agreement, we found that the density of dendritic protrusion decreases in neurons from MAP1B knock-out mice, and the number of filopodia-like protrusions increases when compared with wild type neurons. Finally, the immature phenotype of dendritic protrusions in mature MAP1B-deficient neurons was associated with a reduction in mEPSC amplitude. These findings demonstrate the importance of MAP1B in dendritic spine formation and synaptic maturation.

Actin cytoskeleton plays an important role in dendritic spine morphogenesis and dynamics (68–70). Numerous actin-binding proteins under the control of different pathways, such as Rho-GTPases, regulate actin filament dynamics. MAP1B was recently reported to modulate Rac1-GTPase activity by interacting with Tiam1, a Rac1-GEF. MAP1B-deficient neurons exhibit less Rac1 and cdc42 activity, whereas RhoA activity is increased (18). Here we demonstrate that this alteration is maintained in the adult brain of MAP1B heterozygous mice (MAP1B^+/−), specifically in postsynaptic synaptosome fractions. These animals exhibited decreased Rac1 activity and increased levels of RhoA-GTP. Several findings implicate Rac1 and RhoA in both the formation and maturation of spines. In support of our findings, previous studies reported that a dominant-negative Rac1 causes a reduction in pyramidal neuron spine density in hippocampal slices (26, 52, 54). Neurons expressing this dominant-negative construct have significantly longer dendritic spines than those of controls. Furthermore, Rac1 inhibition reduces spine head growth. Similarly, inhibition of Rac1 function impairs AMPA receptor clustering at synapses and reduces mEPSC amplitude (71). These observations demonstrate a role for Rac1 in spine maintenance and maturation, possibly via its modulation of the actin cytoskeleton (72). In contrast to Rac1, RhoA inhibits spine formation and maturation (53), and thus, it may be inactivated during synaptogenesis. Introduction of constitutive-active RhoA decreases spine density, demonstrating the negative effect of RhoA on spine formation and maintenance (26, 52, 54). Conversely, RhoA inhibition increases the density of spines in cortical and hippocampal mouse neurons (26).

Interactions (direct or indirect) between MAP1B and Tiam1 have already been described (18). In agreement, we found that MAP1B co-immunoprecipitates with Tiam1 in the adult brain. Moreover, we found that MAP1B co-immunoprecipitated with another specific RhoA-GEF, GEF-H1, and that these interactions occurred independently of the presence of tubulin polymers. Interestingly, both Tiam1 and GEF-H1 have been reported to be active in dendritic spines. Tiam1 has been linked to NMDA receptor activity in the development of dendritic arbors and spines, and knockdown of Tiam1 by RNAi in hippocampal neurons leads to a reduction in spine density (28). The RhoA-specific GEF-H1 is an AMPA receptor-interacting protein (73) that translocates into dendritic spines (74). Inhibition of AMPA receptor activity promotes the activation of GEF-H1, which in turn negatively regulates spine development by the activation of the RhoA cascade (73). This GEF has been proposed to mediate the cross-talk between MTs and the actin cytoskeleton (58). RNAi-mediated knockdown of GEF-H1 increases spine density and length, perhaps due to Rac1 inhibition by RhoA (75). Thus, displacing the equilibrium between RhoA activation and inactivation may alter dendritic spine morphology, which could occur if MAP1B loss-of-function is paralleled by an increase in GEF-H1 activity.

The importance of proteins that regulate the actin cytoskeleton is clear, and they are in turn regulated by Rho GTPases during dendritic spine development. We demonstrated that the activity of proteins involved in regulating the morphology of dendritic spines, such as cofilin, is altered in the absence of MAP1B. Cofilin is found at the postsynaptic density (76), where it induces the dynamic disassembly of actin filaments that is required to maintain the length and proper morphology of dendritic spines (77, 78). The decrease in phosphocofilin levels observed in synaptosome fractions from heterozygous mice correlates with a decrease in Rac1 activity. LIM domain kinase (LIMK) is a common downstream effector of Rho family small GTPases (79–81) that phosphorylates and inactivates cofilin (55). LIMK-1 knock-out mice exhibit decreased levels of phosphorylated cofilin (82). Interestingly, spine morphology is also altered in these mice; the spines developing a long neck and small head, similar to those described in the present study. Moreover, the amount of phosphorylated ADF/cofilin was markedly diminished, whereas the total ADF/cofilin remained similar to that observed in wild type neurons (82).

In summary, the present findings support a novel function for MAP1B in controlling dendritic spine morphology and synaptic function via the regulation of actin cytoskeleton through Rac1 and RhoA activity. MAP1B may contribute to the spatiotemporal activation of these Rho-GTPases in dendritic spines by binding to the Rac1 and RhoA GEFs, Tiam1, and GEF-H1.