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Comparative Study
. 2007 Feb 27;104(9):3213-8.
doi: 10.1073/pnas.0611547104. Epub 2007 Feb 20.

Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function

Koji Ikegami  1 , Robb L Heier, Midori Taruishi, Hiroshi Takagi, Masahiro Mukai, Shuichi Shimma, Shu Taira, Ken Hatanaka, Nobuhiro Morone, Ikuko Yao, Patrick K Campbell, Shigeki Yuasa, Carsten Janke, Grant R Macgregor, Mitsutoshi Setou
Affiliations
Comparative Study

Loss of alpha-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function

Koji Ikegami et al. Proc Natl Acad Sci U S A. .

Abstract

Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of alpha- and beta-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular "traffic sign" for motor proteins in neuronal cells. To investigate whether polyglutamylated alpha-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of alpha-tubulin-selective polyglutamylase. In wild-type mice, polyglutamylated alpha-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated alpha-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of alpha-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
PTM of tubulin in ROSA22 mutant mice. (A) Representative Western blot analysis of tubulins in brain lysates of adult wild-type (+/+) and ROSA22 homozygote (−/−) mice. Brain homogenate of ROSA22 homozygote (−/−) mice lacked PGs1 and contained a significantly less amount of polyglutamylated (PG-) and tyrosinated (Tyr-) tubulin than that of wild-type (+/+) mice. The amounts of acetylated (Ac-), α-, β-, γ-tubulin, neurofilament H (NF-H), actin, and GAPDH were not different between wild-type and ROSA22 homozygote. Quantitative analyses of signal intensities are shown in SI Fig. 9. (B and C) Brain lysates from wild-type (+/+) and ROSA22 mutant (−/−) were subjected to high-resolution two-dimensional electrophoresis. (B) The images of CBB-stained two-dimensional gel were colorized and merged; green, wild-type; red, mutant. (C) Major spots were numbered. (D) Mass spectrometry of the α-tubulin carboxyl terminus digested from major spots shown in the illustration in C. (Left) Wild type (W). (Right) Mutant (M). The spot number corresponds to the numbering in the illustration (e.g., W1 corresponds to the sample 1 in the wild type). The peak detected in each spot was ordered numerically, and the corresponding peptide mass and the deduced carboxyl-terminal modifications are listed in SI Table 1. Formally, as indicated in the table, the mass spectrometry cannot exclude the existence of relatively low abundance of biglutamylated forms of Δ2 α-tubulin (Δ2 + 2E). (E) Western blot analysis of high-resolution two-dimensional electrophoresis of tubulins from brains of wild-type and homozygous ROSA22 mice. The blot was probed in sequence with antibodies against polyglutamylated, α-, β-, acetylated, and tyrosinated tubulin.
Fig. 2.
Fig. 2.
Intracellular distribution of polyglutamylated tubulins. (A) SCG explants from wild-type (+/+) and ROSA22 mutant (−/−) were surgically dissected into soma (S) and neurites (N). To obtain equal amounts of tubulin, soma samples contained 3-fold more protein than samples from neurites. The samples were immunoblotted with antibodies specific for polyglutamylated (PG-) or α- or β-tubulin. (B) Hippocampal neuronal cultures from wild-type (+/+) or ROSA22 homozygote mice (−/−) were fixed and stained with mAb B3 that reacted selectively with PG-α-tubulin under the conditions used (SI Fig. 10) together with anti-α-tubulin (mAb, DM1A labeled with FITC) and polyclonal anti-MAP2 antibodies. Both axonal (arrowheads) and dendritic (arrows) processes react with mAb B3. Red, PG-α-tubulin; green, α-tubulin; cyan, MAP2. (Scale bar, 30 μm.) (C) Sagittal sections of brain from control (+/+) and ROSA22 mutant (−/−) mice were stained with B3 or DM1A (green) and TOTO-3 (magenta) to identify DNA. (Scale bar, 20 μm.)
Fig. 3.
Fig. 3.
Effects of α-tubulin polyglutamylation on binding of MAPs to microtubules. (A) Crude microtubules were prepared from brain homogenates of wild-type (+/+) or ROSA22 mutant (−/−) in the presence of ATP or AMP-PNP. Cosedimented kinesins and MAPs were detected by Western blot analysis. The amount of protein applied was monitored by the intensity of the CBB-stained tubulin. (B) Signal intensities were quantified and are represented as a percentage of mutant signal relative to wild-type signal. (Top) ATP-present sample. NA, not applicable. (Middle) AMP-PNP-present sample. (Bottom) Total brain homogenate (bottom). Open columns, wild type; filled columns, mutant. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001, with paired t test.
Fig. 4.
Fig. 4.
Altered distribution of KIF1 in ROSA22 mutant neurons and mice. (A) Equal amounts of soma (S) and neurite (N) lysates prepared from wild-type (+/+) and ROSA22 mutant (−/−) mice were subjected to Western blot analyses, with antibodies against proteins indicated. (B) Signal intensities of bands of KIF1A, 3A, 5, and GAPDH were quantified, and the data are presented as the ratio of the amount in neurites to that in soma. The values presented are mean ± SEM of three independent experiments. ∗∗, P < 0.01 with paired t test. (C) In wild-type (+/+) neurons, EGFP-KIF1A is localized in the soma (white arrow) and entered neurites (small white arrowheads). In neurons from ROSA22 homozygotes (−/−), EGFP-KIF1A was present in the soma (white arrow) but was absent from neurites (white arrowheads). In contrast, EYFP-KIF5A (white arrowheads) was able to enter neurites in neurons from both wild-type and ROSA22 homozygous mice. Green, kinesins; red, MAP2. (Scale bar, 20 μm.) (D) Sagittal brain sections of control (+/+) and ROSA22 mutant (−/−) mice were stained with antibodies that recognize the proteins indicated (green) and TOTO-3 (magenta). Note that the distribution of Tau and MAP1A appears to be altered in the ROSA22 mutant cerebellum. (Scale bar, 20 μm.)
Fig. 5.
Fig. 5.
Mislocalization of synaptic vesicles, cargoes of KIF1A, in ROSA22 mutant mice. (A) Representative example of ultrastructure of the CA1 region of hippocampus in wild-type (+/+) and ROSA22 mutant (−/−) mice. Synaptic vesicles (arrowheads) and the postsynaptic side of the synaptic density (arrow) are indicated. (Scale bar, 500 nm.) (B) The density of synaptic vesicles in synaptic terminals was quantified from three independent mice of each genotype.
Fig. 6.
Fig. 6.
Impaired synaptic transmission in ROSA22 mutant mice. (A) Hippocampal slices were subjected to a brief high-frequency stimulation (100 Hz, 19 pulses). The fEPSP slopes during the entire high-frequency stimulation were recorded from CA1 synapses of wild-type (+/+, black) and ROSA22 mutant (−/−, red) mice. (B) Plot of fEPSP slope against stimulus no. (C) Stimulation no. where fEPSP slopes halved are shown. The data represented are mean ± SEM (n = 8 for wild-type, +/+; n = 9 for mutant, −/−). ∗∗, P < 0.01 with Student's t test. (D) Paired-pulse facilitation (PPF) was quantified by using the ratios of the second to the first fEPSP slopes at interpulse intervals of 50 ms.
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