Evidence for the role of MAP1B in axon formation

Abstract

Cultured neurons obtained from a hypomorphous MAP1B mutant mouse line display a selective and significant inhibition of axon formation that reflects a delay in axon outgrowth and a reduced rate of elongation. This phenomenon is paralleled by decreased microtubule formation and dynamics, which is dramatic at the distal axonal segment, as well as in growth cones, where the more recently assembled microtubule polymer normally predominates. These neurons also have aberrant growth cone formation and increased actin-based protrusive activity. Taken together, this study provides direct evidence showing that by promoting microtubule dynamics and regulating cytoskeletal organization MAP1B has a crucial role in axon formation.

Figure 1

(A) Structure of MAP1B gene. Seven coding exons (1–7) and two noncoding ones (3A and 3U) of the MAP1B protein are shown. The trapping vector insertion occurs immediately after exon 2, which encodes the N-terminal 95 amino acids of the protein. The epitope of antibody 125 used to confirm the genotype of animals is depicted as a straight line. The hatched box represents the microtubule-binding domain (MTBD). (B) Western blot analysis of spinal cord extracts from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) MAP1B mutant mice reacted with a mAb against MAP1B and β-galactosidase (β-Gal). Note that the extracts obtained from the homozygous (−/−) MAP1B contain trace amounts of MAP1B and high levels of β-Gal. Ten micrograms of protein was loaded in each lane.

Figure 2

General morphology and morphometric parameters of control and MAP1B-deficient neurons. (A) Confocal micrograph showing a polarized hippocampal pyramidal neuron from a wild-type animal. The cell that displays one single long axon and several much shorter minor neurites was maintained in culture for 2 d. The cell is double labeled with a mAb against tyrosinated α-tubulin (green) and rhodamine-phalloidin (red). (B) Confocal micrograph showing cultured hippocampal pyramidal neurons from a homozygous (−/−) MAP1B mutant mice. The cells were cultured for 2 d and stained as in B. Note that only one cell is polarized and displays a very short axon-like neurite. Bar, 20 μm. (C) Graph showing the percentage of cells displaying axon-like neurites in cultures from wild-type (▪) and MAP1B-deficient (▨) mice. (D–F) Graphs showing changes in total neuritic length (D), axonal length (E), and total minor neuritic length (F) in hippocampal cell cultures from wild-type (▪) and MAP1B-deficient (▨) mice. Note the significant and selective decrease of axonal length in the MAP1B-deficient neurons. Values represent the mean ± SEM.

Figure 3

High-resolution analysis of control and MAP1B-deficient neurons. (A) High-resolution confocal micrograph showing the distal axonal end of a hippocampal pyramidal neuron from a WT mouse. A low-power micrograph of the neuron is shown in the inset; the arrows indicate the distal axonal segment. (B and C) High-resolution confocal micrographs showing the morphology of cultured hippocampal pyramidal neurons from MAP1B-deficient mice. The cells (A–C) were double labeled with a mAb against tyrosinated α-tubulin (green) and rhodamine-phalloidin (red). Note that MAP1B-deficient neurons display numerous short filopodial extensions and growth cone-like structures around the cell body and along neurites. Bar, 5 μm. (D and E) High-power confocal micrographs showing the morphology of the actin cytoskeleton of axonal growth cones from wild-type (+/+) and MAP1B-deficient (−/−) neurons as reveled by staining with rhodamine-phalloidin. (F and G) Equivalent images to those shown previously but also stained with a mAb against tyrosinated α-tubulin (green). Note the reduction in the size of the growth cone lamellipodial veil and the decrease in the number of radial striations in the mutant neurons. Bar, 5 μm.

Figure 4

Quantitation of microtubule polymer content of control and MAP1B-deficient neurons. (A and B) Quantitative measurements of β-tubulin fluorescence intensity in axons (A) and minor processes (B) from wild-type (▪) and MAP1B-deficient (▨) cultured neurons. For these experiments, cells were extracted with detergents before fixation under microtubule-stabilizing conditions (see MATERIALS AND METHODS). Fluorescence intensity measure ments were performed in the cell body (CB) and along neurites. Within neurites measurements were performed in the initial segment (IS), inner segment (Int), middle segment (Med), external segment (Ext), and growth cones (GC) of axons and minor processes. Note that β-tubulin fluorescence intensity is dramatically reduced in the external axonal segment and in axonal growth cones (C). Equivalent measurements to those shown in A, but from cells extracted with detergents after fixation. Note that there are no significant differences in fluorescence intensity between WT and MAP1B-deficient neurons. A total of 75 cells was analyzed for each experimental condition. Each value represents the mean ± SEM.

Figure 5

Distribution of stable and dynamic microtubules in control and MAP1B-deficient neurons. (A and B) Confocal fluorescence images showing the distribution of tyrosinated (green) and detyrosinated (red) α-tubulin in a cultured hippocampal pyramidal neuron from a wild-type animal. (C) Red-green overlay of the images shown previously. Note that within the axon tyr-tubulin staining predominates at the distal segment (arrow) and growth cone. (D and E) Confocal fluorescence images showing the distribution of tyrosinated (green) and detyrosinated (red) α-tubulin in a cultured hippocampal pyramidal neuron from a MAP1B-deficient mice. (F) Red-green overlay of the images shown previously. Note that although the tyr-immunolabeling decreases along the axon, the one corresponding to detyr-tubulin increases (arrow). For this experiment, cells were fixed with detergents under microtubule-stabilizing conditions before fixation. Bar, 10 μm. (G and H) Quantitative fluorescence measurements of tyrosinated (▪) and detyrosinated (▨) α-tubulin immunolabeling in cytoskeletal preparations from cultured hippocampal pyramidal neurons of wild-type (G) and MAP1B-deficient mice (H). Measurements were performed in equivalent regions to those described in Figure 4. A total of 75 cells was analyzed for each experimental condition.

Figure 6

Quantitation of stable and dynamic microtubules in control and MAP1B-deficient neurons. Quantitative fluorescence measurements of tyrosinated (A) and detyrosinated (B) α-tubulin immunolabeling in cytoskeletal preparations from wild-type (▪) or MAP1B-deficient (▨) cultured hippocampal pyramidal neurons treated with nocodazole for 5 min. Measurements were performed in equivalent regions to those described in Figure 4. (C) Quantitative fluorescence measurements of tyrosinated α-tubulin immunolabeling in cytoskeletal preparations from wild-type (▪) or MAP1B-deficient (▨) cultured hippocampal pyramidal neurons during recovery from nocodazole. For this experiment cultured neurons were treated with nocodazole for 30 min. Fluorescence intensity measurements were performed at the distal axonal segment. A total of 75 cells was analyzed for each experimental condition.

Figure 7

Functional redundancy analysis of control and MAP1B-deficient neurons. Quantitative fluorescence measurements of tau (A) and MAP2 (B) immunolabeling from cultured hippocampal pyramidal neurons of wild-type or MAP1B-deficient neurons. Measurements were performed in cells fixed prior or after detergent extraction performed under microtubule-stabilizing conditions. Regions are equivalent to those of previous figures. Groups: ▪, wild-type neurons fixed before extraction; ▨, wild-type neurons fixed after extraction; □, MAP1B-deficient neurons fixed before extraction; and ▤, MAP1B-deficient neurons fixed after extraction. A total of 75 cells was analyzed for each experimental condition. (C–F) Double fluorescence micrographs of cytoskeletal preparations showing β-tubulin (C and E) and MAP2 (D and F) immunolabeling at the distal axonal third of wild-type (C and D) or MAP1B-deficient (E and F) neurons. Both axons were of equivalent length. Bar, 5 μm.