Microtubule-associated protein 1B (MAP1B) is required for dendritic spine development and synaptic maturation

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.

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.

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.

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).

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).

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.

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.