論文の公開元へCDK5/p35-dependent microtubule reorganization contributes to homeostatic shortening of the axon initial segment
概要
主論文の要旨
CDK5/p35-dependent microtubule reorganization
contributes to homeostatic shortening of
the axon initial segment
CDK5/p35依存的な微小管再編による軸索起始部の構造可塑性機構
名古屋大学大学院医学系研究科
細胞科学講座
総合医学専攻
細胞生理学分野
（指導：久場 博司 教授）
Israt Jahan
【Introduction】
The axon initial segment (AIS) is a highly excitable axonal domain located near the soma
and is involved in generation of action potentials. This excitable nature of AIS is attributed to
its structural characteristics and the accumulation of voltage-gated Na + (Nav) channels, which
occurs through their interaction with a scaffold protein, ankyrinG, and tethering to
submembranous actin-spectrin meshwork at AIS. The structural plasticity of AIS strongly
impacts the output of neurons and plays a fundamental role in physiology and pathology of the
brain. However, the mechanisms linking neuronal activity to structural changes in AIS are not
well understood. Nucleus magnocellularis (NM) is an avian homologue of mammalian
anteroventral cochlear nucleus and well known for shortening of AIS by afferent input during
development. In this study, we examined the mechanisms of this activity dependent AIS
shortening in NM using slice culture of chicken brainstem.
【Materials and Methods】
Chickens (Gallus domesticus) of either sex at embryonic day 11 (E11) were used for
organotypic slice culture, and the effects of pharmacological and genetic manipulations of
signals on AIS were examined in NM by immunohistochemistry and electrophysiology.
【Results】
1) Cultured NM neurons reproduced most features of AIS plasticity in vivo
The length of AIS in NM neurons differed among tonotopic regions after 7DIV (days in
vitro), being slightly shorter for those tuned to high-characteristic frequency sounds, which
was consistent with the observations in vivo (Fig. 1). In this culture, we first tested the
contributions of synaptic activities to AIS length by adding DNQX (20 µM) and TTX (0.1 µM)
to the medium for 3 d from 7DIV. The blockade of spontaneous activity increased the AIS
length, specifically at high-frequency regions, abolishing the tonotopic difference of AIS
length. On the other hand, elevating activity by high-K + treatment (10.6 mM) shortened the
AIS again at the high-frequency regions, increasing the tonotopic difference of AIS length in
NM. Importantly, the AIS shortening by high-K + treatment reduced sodium current and
membrane excitability of neurons at the high-frequency regions. Thus, cultured NM neurons
reproduced most features of AIS plasticity in vivo and should be a good model for examining
the molecular mechanisms of plasticity.
2) Intracellular signals of AIS shortening
We explored the triggers of AIS shortening during high-K + treatment pharmacologically
in neurons at high-frequency regions (Fig. 2). Inhibition of ionotropic glutamate
receptors (iGluRs) with DNQX (20 µM) and AP-5 (50 µM) suppressed AIS shortening.
These receptors cause Ca 2+ influx not only by permeating Ca 2+ but also by activating
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voltage-gated Ca 2+ (Cav) channels via depolarization. Indeed, inhibition of several
subtypes of Cav channels occluded the effects of high-K + treatment, suggesting the
importance of [Ca 2 + ] i elevation in the AIS shortening. AIS shortening was sensitive to
multiple kinase inhibitors, such as KT5720 (0.5 µM) and Rp-cAMPS (100 µM) for
protein kinase A (PKA), GF109203X (50 nM) for protein kinase C (PKC), and TATCN21
(5 µM) for calmodulin-dependent kinase II (CaMKII). Importantly, activation of either
PKA or PKC alone mimicked the effects of high-K + treatment. These results confirmed
the involvement of PKA/PKC/CaMKII in AIS shortening and substantial crosstalk
among the kinases.
3) Activation of CDK5 was required for AIS shortening
The above kinases can activate the extracellular signal-regulated kinase (ERK1/2)
pathway in neurons, and ERK1/2 is known as an upstream molecule of CDK5. Inhibition
of either mitogen-activated protein kinase kinase (MEK1/2) with AZD6244 (10 µM) or
CDK5 with roscovitine (2 µM) suppressed AIS shortening during the high-K + treatment
(Fig. 3). More importantly, these inhibitors occluded AIS shortening during the
activation of either PKA or PKC, supporting the idea that ERK1/2 and CDK5 contribute
to the shortening of AIS downstream of PKA/PKC/CaMKII. We next overexpressed
dominant-negative form of CDK5 (dnCDK5), which occluded the AIS shortening during
the high-K + treatment. Overexpression of CDK5 did not affect the AIS length, whereas
overexpression of CDK5 activator, p35, caused AIS shortening without high-K + treatment
(normal medium). Notably, a mutation in a phosphorylation site of p35(T138A) occluded AIS
shortening. In addition, overexpression of p35 and CDK5 eliminated AIS (34 of 34 cells),
suggesting the importance of CDK5/p35 activity in regulating AIS length.
4) Microtubule reorganization contributed to AIS shortening
We tested the possibility that CDK5 mediates AIS shortening via the disassembly of
microtubules (Fig. 4). We incubated the cultures with microtubule-stabilizing agents, taxol
(50 nM), and taccalonolide AJ (50 nM), and found that these microtubule stabilizers
suppressed the AIS shortening during the high-K + treatment. AIS shortening was also
occluded by tubacin (0.1 µM), an inhibitor of HDAC6, an enzyme that destabilizes
microtubules via deacetylation of tubulin. In addition, taxol occluded AIS shortening
by overexpression of p35 or by the activators of PKA or PKC. Notably, taxol occluded
the elimination of AIS after the overexpression of p35 together with CDK5, consistent
with the idea that the elimination was attributed to the facilitation of AIS shortening rather
than the toxicity of strong CDK5/p35 signals. Moreover, the inhibition of PP1/PP2A by
okadaic acid, which promotes phosphorylation of p35 at T138, shortened AIS and this
AIS shortening was suppressed by taxol, suggesting that phosphorylation of p35 at T138
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underlies AIS shortening via interaction with microtubules. In contrast, in neurons at lowfrequency regions, overexpression of p35 did not affect AIS length.
【Discussion】
We found that activation of CDK5 is critical for AIS shortening in NM. It is important to
note that CDK5 activity promoted AIS shortening to a different extent among tonotopic
regions, with the effects being more prominent at high-frequency regions, which mediated the
tonotopic difference of AIS length in NM. CDK5/p35 may regulate microtubule remodeling
in pleiotropic manner depending on autophosphorylation. CDK5 phosphorylates p35 at S8,
allowing its translocation from the plasma membrane for microtubule polymerization.
However, CDK5 also phosphorylates p35 at T138, preventing this interaction and inhibiting
microtubule polymerization, while T138 is dephosphorylated by phosphatases. Therefore, one
possible explanation for the difference of AIS length among tonotopic regions is that the levels
of these phosphatases differ within NM, being lower at high-frequency regions, suppressing
microtubule polymerization and facilitating AIS shortening.
【Conclusion】
In this study, we showed in organotypic cultures of NM that activity dependent AIS
shortening occurs through disassembly of microtubules at distal AIS via activation of
CDK5/p35 signals. This study emphasizes the importance of microtubule reorganization and
regulation of CDK5 activity in structural AIS plasticity and tonotopic differentiation of AIS
structures in the brainstem auditory circuit.
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Figure 1 Activity-dependent AIS shortening in NM neurons
A, Development of an avian auditory system in vivo and in vitro. B, Brainstem auditory circuit of chickens. AN,
Auditory nerve; NL, Nucleus laminaris. C, NM is tonotopically organized along the rostromedial-caudolateral axis. In
most rostral and caudal slices, NM was defined as high- and low-frequency regions, respectively. R, rostral; L, lateral.
D, AIS immunostained with panNav antibody (green, arrowheads) after visualizing NM neurons (TMR, magenta) at
10DIV. E, Length of AIS. Values from individual cells are plotted (open circles) in this and subsequent figures.
Numbers in parentheses indicate the number of cells. F–H, Synaptic and spike activity was blocked by DNQX/TTX
for 7–10DIV. Time course of experiment (F), AIS (green) of NM neurons (magenta) (G), length (H) of AIS. I–K,
Membrane was depolarized by increasing [K+]o in the culture medium by two times (10.6 mM, 2x[K+] medium) for
7–10DIV. Time course of experiments (I), AIS of NM neurons (J), length (K) of AIS. * p < 0.05, ** p < 0.01 between
tonotopic regions by Kruskal-Wallis test.
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Figure 2 AIS shortening occurred via activation of MEK and CDK5 pathways
A, Time course of experiments. Slices containing high-frequency regions of NM were incubated with inhibitors in a
2x[K+] medium or with activators in a normal (1x[K+]) medium for 7–10DIV. B, Schematic drawing of Ca2+ sources
in NM neurons. C, AIS of NM neurons. C, Length of AIS. The numbers in parentheses indicate the number of cells.
D, MEK signaling pathway. E, Effects of kinase inhibitors on AIS length in the 2x[K+] medium. F, Activators of PKA
and PKC shortened AIS in the 1x[K+] medium. Numbers in parentheses indicate the number of cells. ** p < 0.01
compared with 2x[K+] (C, E) or with 1x[K+] (F) by one-way ANOVA and post-hoc test.
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Figure 3 AIS shortening occurred in a manner dependent on CDK5 activity
A, Time course of experiments. Plasmids were introduced into NM neurons at E2, Slices were prepared at E11 and
DOX was added to the culture medium at 6–10DIV. B, tdTomato (red) was expressed in NM neurons (ipsi) in slice
culture stained with panNav antibody (white). Dotted line indicates the midline. C–H, AIS of NM neurons with (ipsi)
or without (contra) overexpression of dnCDK5 in 2x[K+] medium (C), and of p35 (E), or p35(T138A) (G) in normal
(1x[K+]) medium. Plasmids used are shown in each panel. Intensity profiles of Nav signal are the average of 10 cells
(D, F, H). I, J, Length of AIS in 2x[K+] (I) and 1x[K+] (J) media. Numbers in parentheses indicate the number of cells.
* p < 0.05, ** p < 0.01 compared with mock by Student’s t-test (I) and one-way ANOVA and post-hoc test (J).
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Figure 4 CDK5 mediated AIS shortening via reorganization of microtubules
A, Time course of experiments. HCF slices were incubated with stabilizers of microtubules (MT) or actin during
treatment with 2x[K+] medium, FSK or PMA, or okadaic acid for 7–10DIV. B, Pharmacological manipulation of MT
dynamics. C, Effects of MT and actin filament stabilizers on AIS of NM neurons in 2x[K+] medium. D, E, MT
stabilizers occluded AIS shortening by 2x[K+] medium (D, left), by FSK, PMA (D, right), or by okadaic acid (E).
F–H, Taxol occluded AIS shortening by overexpression of p35 (G). Time course of experiments (F) and AIS length
(H). Numbers in parentheses indicate the number of cells. ** p < 0.01 compared with 2x[K+] (D, left), 1x[K+] (E) by
one-way ANOVA and post-hoc test, without Taxol (D, right) by Kruskal-Wallis test, or without Taxol (H) by Student’s
t-test.
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