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First published online 27 March 2007
doi: 10.1242/jcs.001461
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Research Article |
1 Mitsubishi Kagaku Institute of Life Sciences (MITILS), 11 Minamiooya, Machida, Tokyo 194-8511, Japan
2 Laboratory of Metabolic Regulation Research, Kyushu University Bio-Architecture Center, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan
3 Graduate School of Environment and Information Sciences, Yokohama National University, Yokohama 240-8501, Japan
4 CREST, JST, 4-1-8 Honcho, Kawaguchi 332-0012, Japan
* Author for correspondence (e-mail: kaoru{at}mitils.jp)
Accepted 26 February 2007
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Summary |
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 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
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Key words: Calcium signal, Dendritic arborization, Development, Microtubule dynamics, Phosphorylation, Stathmin
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Introduction |
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TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
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). Thus, dendrites act not only as interfaces for neuronal inputs but also as modulators of neuronal information. Consequently, precise dendritic development is an important neuronal process for the construction of neural circuits that function properly.
The dendritic morphology of the cerebellar Purkinje cell (PC) is the most complicated among the various neuronal cell types. The PC dendritic tree extends directly towards the pial surface and branches during the second and third postnatal weeks in rodents (Altman, 1972; Weiss and Pysh, 1978). Total dendritic field and branch length peak around the end of this period. During dendritic arborization, granule cell precursors migrate into the internal granule cell layer through the PC layer, and the migrating granule cell axon (called a parallel fiber) extends laterally in the molecular layer, where it contacts PC dendrites (Wong and Ghosh, 2002). Each mature PC forms 
106 synapses with the parallel fibers. Neural activity regulates the development of PC dendrites (Altman, 1972; Rakic, 1975; Wong and Ghosh, 2002).
Intracellular calcium signaling is involved in the activity-dependent regulation of development and patterning of dendritic morphology (Lohmann et al., 2002; Redmond et al., 2002; Schilling et al., 1991; Wong and Ghosh, 2002). Calcium/calmodulin-dependent protein kinase (CaMK) II and CaMKIV mediate calcium-dependent dendritic growth (Fink et al., 2003; Gaudilliere et al., 2004; Redmond et al., 2002; Wu and Cline, 1998). CaMKII localizes at postsynaptic sites, where it functions as a regulator of dendritic arborization in various neural cell types (Wong and Ghosh, 2002), and its kinase activity is regulated by neural activity. However, the specific molecular mechanisms that underlie the activity-dependent modulation of dendritic architecture remain to be elucidated.
Microtubules (MTs) are essential structural components of dendrites, and neural activity-dependent dendritic formation is mediated in part by the modulation of MT stability in a CaMKII-dependent manner (Vaillant et al., 2002). Among the factors regulating MT stability, stathmin is a candidate molecule that regulates MT dynamics in response to calcium signaling. Stathmin destabilizes the microtubule in two distinct ways, by sequestering 
- and 
-tubulin heterodimers and by promoting MT catastrophe (Cassimeris, 2002; Grenningloh et al., 2004). These MT-destabilizing activities are suppressed by phosphorylation of stathmin at Ser16 and Ser63 (Cassimeris, 2002; Grenningloh et al., 2004; Wittmann et al., 2004), and CaMKII phosphorylates stathmin at Ser16 (le Gouvello et al., 1998).
Other stathmin family members, SCG10, SCLIP and RB3, also bind to tubulin and regulate MT dynamics (Charbaut et al., 2001). These molecules are expressed in the central nervous system (Ozon et al., 1999; Sugiura and Mori, 1995) and share a C-terminal interaction domain that contains a tubulin-binding region (predicted -helix structure) and an N-terminal regulatory region containing phosphorylation sites as stathmin (Charbaut et al., 2001; Gavet et al., 1998) (Fig. 1A). A major difference between stathmin and the other members is that stathmin is distributed in the cytosol, whereas the other three members localize on Golgi and vesicle membranes (Charbaut et al., 2005; Gavet et al., 1998). Notably, SCG10 is phosphorylated by CaMKII in vitro (Antonsson et al., 1997). These results suggest that stathmin and related proteins, especially SCG10, mediate calcium signal-dependent dendritic arborization during postnatal development by regulating MT dynamics.
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). In particular, no obvious abnormalities of neuronal morphology are observed in the pyramidal cells of the lateral amygdala and hippocampal CA1 region of the knockout mice (Shumyatsky et al., 2005). However, stathmin depletion with antisense oligonucleotides prevents nerve growth factor-stimulated neurite outgrowth in rat PC12 cells (Di Paolo et al., 1996). Therefore, it remains to be determined whether stathmin is an essential regulator of normal dendritic development. Here, we demonstrate that stathmin is a regulator of dendrite arborization, the activity of which is downregulated during dendritic development by two distinct mechanisms: reduction in gene expression and phosphorylation. Importantly, proper downregulation of its activity is critical for normal dendritic development. The appropriately controlled stathmin activity and the resulting MT stability in PCs are important for dendritic arborization.
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Results |
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; Weiss and Pysh, 1978). To identify molecules that regulate the dynamics of the dendritic tubulin cytoskeleton, we prepared PCs from frozen brain sections of P12 and P15 mice by the laser capture microdissection method (Fig. 1B). PC-specific RT-PCR-differential display identified 23 cDNAs whose mRNA levels in PC differed at the two ages. Among those that were downregulated during this period, we focused on stathmin (Fig. 1C) because it can destabilize MTs. We then used in situ hybridization to quantify the developmental decrease of the stathmin mRNA level in the PC soma (P12, 100±1.9%; P15, 72.6±2.1%; P18, 64.1±1.8%; Fig. 1D,E). An immunohistochemical analysis showed that stathmin protein in the cerebellar cortex, including the PC somata, also decreased dramatically from P12 to P18 (Fig. 1F). In the molecular layer, the strong signals in axonal fibers of granule cells migrating from the external granule cell layer to the inner granule cell layer decreased around the end of the migration, at P18. We also analyzed the developmental fluctuation of stathmin protein in cerebellum (Fig. 1G). The stathmin protein level normalized to -tubulin levels decreased from P12 to P18 (P12, 1.00±0.29; P15, 0.29±0.08; P18, 0.11±0.04; Fig. 1H).
Extension of stabilized MTs in PC dendrites is inversely correlated with stathmin expression
We next examined changes in the stability of dendritic MTs during the period of dramatic dendritic growth. MAP2 and acetylated -tubulin are markers for stable MTs (LeDizet and Piperno, 1986; Matus, 1994; Piperno et al., 1987). MAP2, a major MT-associated protein in dendrites, stabilizes MTs and is required for proper dendritic growth (Goedert et al., 1991; Harada et al., 2002; Matus, 1994). Immunohistochemical analysis revealed that the length of continuous immunoreactivity (signal length) for MAP2 and acetylated -tubulin in the molecular layer increased from P9 to P18 (Fig. 2A). These increases were inversely correlated with the decrease in the stathmin mRNA levels (Fig. 2B).
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) in primary cultures of PCs and asked whether dendritic extension was suppressed. In this system, cultured PCs reproduce several morphological and electrophysiological changes seen in intact postnatal postnatal rodents with similar time-courses (Tabata et al., 2000). We prepared three cDNA constructs encoding Myc-tagged stathmin (Sta), a constitutively active form of stathmin (4A), and GFP, respectively (Fig. 3A; Table 1). Four serine residues of stathmin (Ser16, Ser25, Ser38 and Ser63) are phosphorylated by various kinases, which reduces the MT-destabilizing activity (Cassimeris, 2002; Wittmann et al., 2004). The 4A mutant stathmin, in which all four serine residues are replaced by alanine residues, has constitutive MT-destabilizing activity regardless of extracellular stimuli (Gavet et al., 1998).
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The expression vectors were transfected into PCs at DIV-0. Transfected PCs were identified at DIV-14 by staining with anti-calbindin and anti-Myc antibodies (to visualize Sta or 4A) or by staining with an anti-calbindin antibody and observing GFP fluorescence (GFP). Compared with the GFP-expressing PCs, which showed normal dendritic morphology, Sta-transfected PCs showed decreased dendritic development (Fig. 3B). Transfection of the 4A construct resulted in a more severe phenotype with much less dendritic development (Fig. 3B). Overexpression of stathmin significantly diminished the total dendritic length (GFP, 513±33 µm; Sta, 178±30 µm; 4A, 55±14 µm; Fig. 3D) and the number of primary dendrites (GFP, 3.62±0.23; Sta, 1.88±0.25; 4A, 1.00±0.21; Fig. 3E).
Phosphorylation of stathmin at Ser16 is regulated by CaMKII in developing PCs
Among the four phosphorylation sites, phosphorylation specifically at Ser16 and Ser63 inhibits the MT-destabilizing activity (Cassimeris, 2002; Lawler, 1998; Wittmann et al., 2004). We performed immunoblotting of cerebellar extracts prepared from P12, P15 and P18 mice with an antibody against phospho-Ser16 (Fig. 1I). The phosphorylation signal of stathmin at Ser16 increased from P12 to P15 and then decreased slightly at P18 (P12, 1.00±0.08; P15, 2.29±0.19; P18, 1.71±0.38; Fig. 1J) in spite of the decreased stathmin level from P12 to P15 (Fig. 1G,H).
In T lymphocytes, the Ser16 of stathmin is a target of CaMKII (le Gouvello et al., 1998). In our culture system, PC dendrites begin to develop at DIV-7. When the PC culture was treated with a CaMKII inhibitor, KN-93, from DIV-7 to DIV-9, autophosphorylation of CaMKII at Thr286 was inhibited (data not shown), and the phosphorylation level at Ser16 of stathmin was significantly reduced (Fig. 4A,B), indicating that phosphorylation at Ser16 is also regulated by CaMKII in developing PCs in culture. A similar result was obtained with PCs treated with tetrodotoxin (TTX), which blocks voltage-dependent Na+ channels and inhibits depolarization of neurons (Fig. 4A,B). TTX treatment also reduced the CaMKII phospho-Thr286 signal in PCs (data not shown). These results strongly suggest that CaMKII phosphorylates Ser16 in a neural activity-dependent manner.
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Because N-methyl-D-aspartate (NMDA) receptor-mediated excitatory postsynaptic current (EPSCs) are not detected in PCs from P9 to P25 (Aiba et al., 1994; Kakizawa et al., 2000; Kano et al., 1995), CaMKII activity appears to be regulated in PCs by calcium entry from the extracellular space via L-type calcium channels or from the endoplasmic reticulum via inositol 1,4,5-trisphosphate receptor type 1 [Ins(1,4,5)P3R1]. The channel activity of Ins(1,4,5)P3R1 is positively regulated by mGluR1 (Nakanishi, 1994). Blockade of either L-type calcium channels by nifedipine or mGluR1 by CPCCOEt caused a significant reduction in the level of phosphorylation at Ser16 (Fig. 4C-F). The reduction of phosphorylation level was not due to the reduced stathmin level, because KN-93, TTX, nifedipine or CPCCOEt treatment did not result in any obvious change in stathmin signal levels in PCs (Fig. 4G,H).
The effect of stathmin on dendritic arborization is regulated by CaMKII
To test whether phosphorylation of stathmin by CaMKII affects dendrite development, cultured PCs were treated with KN-93 from DIV-7 to DIV-14. KN-93 treatment limited dendrite development (Fig. 3C), decreasing the total length of the dendritic arbor of GFP-expressing PCs to175±14 µm, one third of that of non-treated PCs (Fig. 3B,D). KN-93 also significantly decreased the number of primary dendrites (Fig. 3B,E). These data indicate that CaMKII is a positive regulator of the dendritic arborization of PCs.
From the above results, we hypothesized that CaMKII exerts its effect on dendritic arborization through the phosphorylation of stathmin at Ser16. To test this directly, we constructed S16A and S16E, in which Ser16 was altered to alanine and glutamate, respectively (Table 1). The S16E mimics Ser16 phosphorylation because stathmin containing the Ser to Glu mutation in the phosphorylation sites shows a similar interaction with tubulin as artificially phosphorylated stathmin (Niethammer et al., 2004). Dendrites of S16A-transfected PCs showed extremely abnormal morphology compared to the GFP-transfected PCs (Fig. 3B,D,E). The total length of dendritic branches (59±19 µm) was one third of that of Sta-transfected PCs (Fig. 3D) and was comparable to that following KN-93 treatment. The PCs transfected with S16E were similar to those transfected with Sta in dendritic arborization (total dendritic length: 229±36 µm; number of primary dendrites: 2.52±0.39; Fig. 3B,D,E).
Stathmin knockdown significantly reduces dendritic extension in cultured PCs
To clarify the role of endogenous stathmin in dendritic extension in cultured PCs, we performed RNA interference experiments. Fig. 5 shows the efficacy of siRNAs transfected into rat PC12 cells in knocking down stathmin expression. Anti-stathmin siRNA-2, but not siRNA-1, significantly decreased the stathmin level (ratio of control siRNA to siRNA-1 to siRNA-2=1 to 0.70±0.24 to 0.25±0.08; control siRNA versus siRNA-1, P<0.28; control siRNA versus siRNA-2, P<0.001; Student's t-test) (Fig. 5A,B). We then introduced Alexa Fluor-546-conjugated control siRNA and stathmin siRNA-2 into cultured PCs at DIV-0, and observed the dendritic extension of transfectants at DIV-14. The RNA interference activity of synthetic siRNAs persists for at least 3 weeks in mammalian cultured neurons (Omi et al., 2004). A quantitative analysis revealed that stathmin siRNA-2 significantly reduced the total dendritic length by 25% (control siRNA, 537±36 µm; anti-stathmin siRNA-2, 400±54 µm; Fig. 5C,D).
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). In PC somata at DIV-9, the intracellular distribution of SCG10 was mainly restricted to the perinuclear Golgi apparatus, whereas stathmin was distributed throughout the cytoplasm (Fig. 6A). Similar distributions were observed in PC somata at DIV-14 (data not shown). Exogenously expressed SCG10 was also distributed mainly in the perinuclear region of PCs at DIV-14 (Fig. 6B). Overexpression of SCG10-Myc in cultured PCs significantly limited the dendritic extension compared with GFP overexpression (GFP, 542±35 µm; SCG10, 252±40 µm; Fig. 6C,D), although to a lesser degree than stathmin (Fig. 3; Sta/GFP=34.7%; SCG10/GFP=46.5%).
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The amino acid sequences surrounding Ser50 of SCG10 and Ser16 of stathmin are quite similar (Antonsson et al., 1997), suggesting that SCG10 activity may be regulated by phosphorylation of Ser50 by CaMKII. We constructed a S50A SCG10 mutant in which Ser50 was replaced by Ala. Introduction of S50A into PCs significantly reduced dendritic extension (S50A, 224±36 µm; Fig. 6C,D), but there was no difference in the total dendritic length between PCs transfected with SCG10 and S50A (P>0.60, Student's t-test).
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Discussion |
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TopSummaryIntroductionResultsDiscussionMaterials and MethodsReferences |
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; Sugiura and Mori, 1995).
Stathmin knockout mice show normal neuronal morphology (Shumyatsky et al., 2005), suggesting that stathmin-related proteins, such as SCG10, make up for the depleted stathmin activity in the developing neurons. Indeed, the stathmin-related proteins SCG10, SCLIP and RB3 are expressed at an early stage of brain development (Ozon et al., 1998). However, our RNA interference experiment showed that dendritic length is slightly but significantly reduced by knockdown of endogenous stathmin (Fig. 5). Precise quantitative analysis of dendritic arborization in the knockout mice was not performed by Shumyatsky et al. By contrast, our analysis of the effects of knockdown in cultured cells was sensitive enough to detect the slight but significant effect on the dendritic morphology. It is consistent with the result that stathmin depletion with antisense oligonucleotides prevents nerve growth factor-stimulated neurite outgrowth in rat PC12 cells (Di Paolo et al., 1996). Taken together these results indicate that stathmin is indeed involved in the regulation of dendritic arborization.
Stathmin overexpression limited the dendritic growth of cultured PCs, a phenotype that was more dramatic with the expression of the 4A mutant stathmin, which has constitutive MT-destabilization activity. These results clearly demonstrate that hyperactivity of stathmin reduces dendritic arborization. In developing oligodendrocyte progenitors, high levels of stathmin result in the maintenance of oligodendrocytes with a simple morphology and fewer branches than normal oligodendrocytes (Liu et al., 2005). Thus, both in neurons and glial cells, downregulation of stathmin is crucial for the formation of complex processes.
Knockdown and overexpression of stathmin produced similar effects on the dendritic arborization of PCs. We suggest that knockdown of endogenous stathmin leads to increased MT stability, and consequently to decreased dendritic elongation (Grenningloh et al., 2004). Indeed, microinjection of anti-stathmin antibody, which inhibits tubulin binding to stathmin, significantly decreases plus end elongation velocity of MT in newt lung cells (Howell et al., 1999). Moreover, pharmacological treatment with drugs that stabilize MTs inhibits neurite outgrowth (Letourneau and Ressler, 1984). A properly regulated balance between polymerization and depolymerization of MT appears to be critical for the normal elongation of dendritic arbors. The balance of MT states may be regulated by precisely optimized stathmin activity.
Our results indicate that the phosphorylation of stathmin at Ser16 is positively regulated by CaMKII in a neural activity-dependent manner. Overexpression of the stathmin S16A mutant in PCs led to severe impairment of dendritic growth that was equivalent to the effect of the 4A mutant. This clearly demonstrates that phosphorylation at Ser16 is essential for the downregulation of stathmin activity that is critical for proper dendritic development. Indeed, the phosphorylation level at Ser16 dramatically increased during the PC development (relative ratio of phospo-Ser16 per stathmin; P12, 1.00; P15, 7.90; P18, 15.5; from Fig. 1H,J). The construct with partial constitutively negative activity (S16E), however, was not clearly different from the wild-type Sta construct, suggesting that phosphorylation at another serine residue, perhaps Ser63, is also involved in this process. Taken together, we suggest that a decrement of stathmin expression and suppression of the remaining stathmin by CaMKII phosphorylation stabilizes dendritic MTs during dendritic development.
Stathmin mediates calcium signal-dependent development of dendritic arborization
Intracellular calcium signaling mediated by CaMKII and CaMKIV plays an important role in the regulation of dendritic growth during development (Fink et al., 2003; Gaudilliere et al., 2004; Redmond et al., 2002; Vaillant et al., 2002; Wu and Cline, 1998). CaMKIV acts through a transcription-dependent mechanism, in which CaMKIV activation triggers dendritic arborization that is dependent on the transcription factor CREB (Redmond et al., 2002). By contrast, CaMKII phosphorylates MAP2, which increases the association of MAP2 with MTs. This stabilizes the MTs and leads to reversible dendrite formation (Vaillant et al., 2002). In addition to the CaMKII-MAP2 pathway, we suggest that CaMKII modulates dendritic MT stability through stathmin phosphorylation. Thus, stathmin and MAP2 may both mediate the calcium-CaMKII regulation of dendritic MT stability that leads to the development of dendritic arborization.
Voltage-gated calcium channels (VGCCs) and mGluR1 trigger the calcium signal required for the proper regulation of dendrite formation in PCs. For example, VGCC activation leads to an increase in dendritic growth of cultured neurons (Gaudilliere et al., 2004; Redmond et al., 2002), blockade of group 1 mGluRs (mGluR1 and mGluR5) inhibits the arborization of the dendritic tree of PCs (Catania et al., 2001), and the PCs of mGluR1 knockout mice have abnormal dendritic morphology (Aiba et al., 1994). Here we showed that stathmin phosphorylation at Ser16 is positively regulated by VGCCs and mGluR1 in PCs. Because phosphorylation at Ser16 is critical for normal dendritic development, it follows that both VGCC and mGluR1 mediate the neural activity-dependent dendrite formation that depends on the proper regulation of stathmin activity. Therefore, we suggest that calcium elevation, either from the extrasynaptic space or from intracellular calcium stores, activates CaMKII, which in turn phosphorylates stathmin at Ser16 to stabilize dendritic MTs.
SCG10 regulates dendritic arborization through a different mechanism from stathmin
Overexpression of a membrane-anchored member of the stathmin family, SCG10, reduced the dendritic branch length of PCs, as was observed for stathmin (Fig. 6). However, overexpression of S50A had the same effect as the wild-type SCG10 on the limitation of dendritic extension (Fig. 6). These data suggest that SCG10 modulates dendrite arborization independently of CaMKII regulation. Ser62 and Ser73 of SCG10 are phosphorylated by JNK1 (Tararuk et al., 2006), and the neurite length of cultured cortical neurons is reduced by overexpression of SCG10 S62A/S73A, a JNK1-phosphorylation-site mutant (Tararuk et al., 2006). Moreover, intraperitoneal injection of kainate upregulates the phosphorylation level of SCG10 at Ser73 in the hippocampus, whereas phosphorylation at Ser50 is constitutive (Morii et al., 2006). Together, these results strongly suggest that stathmin and SCG10 mediate distinct signaling cascades, CaMKII and JNK1, respectively, in the regulation of dendritic arborization. This may be due in part to their different intracellular distribution.
Stathmin may be involved in stabilizing dendritic morphology in response to calcium signals triggered by neuronal contact during neural network formation
It has been proposed that calcium-dependent transcription modulates overall growth of the dendritic tree (Wong and Ghosh, 2002). New dendritic branches are added to the preexisting dendrite in a transcription-dependent manner. Local calcium elevation in the dendrites, however, may regulate the stability of the dendritic branch (Wong and Ghosh, 2002). We propose that once a new dendritic segment makes synaptic contacts with an axon terminal, local calcium signaling, triggered by VGCC and mGluR activation, stabilizes the dendritic MT cytoskeleton through the CaMKII-stathmin pathway, which then leads to the stabilization of new dendritic branches. In a continuous process, dendrites extend from the tip of the stabilized branch in a transcription-dependent manner, followed by MT stabilization in response to synaptic inputs. These processes appear to have an important role in specifying the morphology of the dendritic tree.
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Materials and Methods |
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-tubulin antibody B-5-1-2 and mouse anti-acetylated -tubulin antibody 6-11B-1, Sigma-Aldrich (St Louis, MO); rabbit anti-stathmin antibody O0138, Sigma-Aldrich, and 569391, Calbiochem (San Diego, CA); rabbit anti-phospho-stathmin (Ser16) antibody and mouse anti-Myc antibody 9E10, Santa Cruz Biotechnology, Inc (Santa Cruz, CA); goat anti-SCG10 antibody ab21190, Abcam (Cambridge, UK); and rabbit and mouse anti-calbindin antibodies, Chemicon and Swant Swiss Antibodies (Bellinzona, Swiss), respectively. DRAQ5 (Biostatus Ltd, Leicestershire, UK), which fluoresces in the far red, was used for nuclear staining.
Plasmid construction
A mouse stathmin cDNA that was Myc-epitope-tagged at the C terminus (stathmin-Myc) was obtained by PCR using a 5' primer (5'-GACATGGCATCTTCTGATATTCAGGTGAAAGAGCTGG-3'), a Myc-tag-encoding 3' primer (5'-TTACAGGTCTTCCTCACTGATCAGCTTCTGTTCCTCGTCAGCCTCAGTCTCATCCGCGGGGTCTTTGG-3'), and dT-primed cDNAs prepared from mouse brain as templates. A SCG10-Myc cDNA was similarly obtained using the 5' primer 5'-ACAATGGCTAAAACAGCAATGGCCTACAAGG-3' and the 3' primer 5'-TTACAGGTCTTCCTCACTGATCAGCTTCTGTTCCTCGCCAGACAGTTCAACCTGCAGTTCCTTGTTCC-3'. PCR mutagenesis of Ser to Ala (GCG or GCC) or Glu (GAG) was carried out using an overlap-extension reaction (Higuchi et al., 1988) with the stathmin-Myc or SCG10-Myc cDNA as a template. PCR products were ligated into the cloning vector pCRII-TOPO (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.
To generate a PC-specific stathmin-Myc or SCG10-Myc expression plasmid (CMV-L7-stathmin-Myc or CMV-L7-SCG10-Myc), stathmin-Myc or SCG10-Myc was introduced into exon 4 of the L7 gene cassette (Ichise et al., 2000; Oberdick et al., 1990), and then the L7-stathmin-Myc fragment was subcloned into the NotI site of pcDNA3 (Invitrogen). A PC-specific GFP expression vector was generated by inserting a 1.3-kb PCR fragment of the L7 promoter sequence downstream of the 0.6-kb CMV promoter in the pcDNA3.1/CT-GFP-TOPO vector (Invitrogen). The PCR fragment amplified from mouse genomic DNA with the 5' L7A primer (Smeyne et al., 1995) and the 3' primer 5'-CAGCCTGCAAGGAACAGCGCTGCT-3' contained approximately 1 kb of the promoter, the first and second exons, and the first intron of the L7 gene (Fig. 3A). As a result of these manipulations, the first ten amino acids of the L7 protein, including the initiator Met with an additional Gly residue generated by the cloning strategy, were fused in frame to the N terminus of GFP. The L7 promoter in this CMV promoter cassette directed expression of stathmin-Myc or GFP specifically and efficiently to PCs under in vitro culture conditions.
Animals and brain sections
All procedures involving the use of animals complied with the guidelines of the National Institute of Health and were approved by the Animal Care and Use Committee of Mitsubishi Kagaku Institute of Life Sciences (MITILS). C57BL/6 mice were purchased from CLEA Japan Inc. (Tokyo, Japan). For preparation of frozen brain sections, mice were sacrificed, and their brains were dissected and immediately frozen on dry ice. Cryosections (10-µm thick) were air-dried and stored at –80°C until use for in situ hybridization and immunohistochemistry.
Laser capture microdissection and PCR-differential display
Mice were sacrificed at postnatal day (P)12 or P15, and the brains were immediately frozen on dry ice powder. Sagittal cryosections, cut at 30-µm thickness, were fixed with 70% ethanol, dehydrated with ethanol, immersed in xylene, and then air-dried. The PC layers were collected using the Laser Capture Microdissection System (LM2000, Arcturus Eng. Inc., Mountain View, CA). Total cellular RNA was extracted from the collected PCs using Sepasol-RNA I (Nacalai Tesque, Kyoto, Japan). PCR-differential display was performed as described previously (Matsuo et al., 2000). One of the candidate genes was amplified with an arbitrary primer (5'-TCTGTGCTGG-3
) and a T12GC anchor primer. DNA sequence analysis and a database search revealed that it included nucleotides 782-963 of mouse stathmin 1 (GenBank accession number: NM019641).
In situ hybridization
The stathmin cDNA obtained by PCR-differential display was amplified by PCR and subcloned into the vector pCRII-TOPO. The vector was digested with SpeI or EcoRV to generate a template for the in vitro transcription of an antisense or sense cRNA probe, respectively. Digoxigenin-labeled cRNA probes were produced by transcription with T7 or Sp6 RNA polymerase. In situ hybridization was performed as described previously (Matsuo et al., 2000) except for the hybridization temperature and the washes after hybridization. Hybridization was performed at 37°C. Sections were washed with 4xSSC (0.6 M NaCl, 0.06 M sodium citrate, pH 7.0) at 37°C for 30 minutes and then at 42°C for 30 minutes prior to RNase A treatment. Sections were washed with 2xSSC for 30 minutes and 0.5xSSC at 42°C for 30 minutes following the RNase A treatment.
Immunohistochemistry
Brain sections were fixed with 10% formaldehyde neutral buffer (pH 7.0; Nacalai Tesque, Kyoto, Japan) at room temperature (RT) for 30 minutes. After being washed with phosphate-buffered saline (PBS), the sections were treated with PBST (PBS supplemented with 0.1% or 0.2% Triton X-100) three times at RT for 3 minutes each, followed by three washes with PBS for 5 minutes each. The sections were then treated with blocking buffer [2% bovine serum albumin (BSA) in PBS] at RT for 1 hour. Reactions with primary antibodies were performed in blocking buffer containing mouse anti-MAP2 antibody (1:250) or mouse anti-acetylated -tubulin antibody (1:100) at 4°C overnight. After three washes with PBS for 10 minutes each, the sections were incubated with FITC-conjugated secondary antibodies at 4°C overnight. Sections were then washed with PBS three times for 10 minutes each, and mounted with ProLong Gold antifade reagents (Molecular Probes, Eugene, OR). The fluorescent signals were examined with a laser-scanning confocal microscope (LSM 5 PASCAL, Carl Zeiss, Jena, Germany). For immunohistochemistry of stathmin and calbindin, mice were perfused with 4% paraformaldehyde (PFA), and the brains were removed. After postfixation in 4% PFA, the brains were kept in 25% sucrose at 4°C overnight. Cryosections of 14-µm thickness were permeabilized by PBST treatment, washed, and blocked with blocking buffer. A rabbit anti-stathmin antibody (Calbiochem, 1:500) and a mouse anti-calbindin antibody (1:500) were used as primary antibodies.
Preparation of whole cell extract from cerebellum
Mice were sacrificed at P12, P15 and P18, and dissected cerebellums were immediately frozen with liquid nitrogen. The cerebellums were homogenized with TNE buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl and 2 mM EDTA) containing 1x protease inhibitor cocktail (Sigma) and 1% NP-40. After measurement of protein concentration using the BCA protein assay kit (Pierce, Rockford, IL), the whole cell lysates were subjected to polyacrylamide gel electrophoresis and immunoblotting.
RNA interference
Alexa Fluor-546-labeled negative-control and anti-rat stathmin siRNA duplexes were designed and synthesized by Qiagen (Valencia, CA). The stathmin target sequences used were 5'-CUGGAGGAAAUUCAGAAGAAA-3' (stathmin siRNA-1) and 5'-CUGUACUGGAAUGGUUAAUAA-3' (stathmin siRNA-2). PC12 cells were plated in a 6-well plate 24 hours before transfection, then transfected with siRNA (final concentration 100 nM) using Lipofectamine 2000 (Gibco, Rockville, MD). Three days after the transfection, cells were collected and lysed in SDS sample buffer for SDS polyacrylamide gel electrophoresis and immunoblotting.
Immunoblotting
A rabbit anti-stathmin antibody (Calbiochem, 1:5000), a rabbit anti-phospho-stathmin (Ser16) antibody (Santa Cruz Biotechnology, Inc, 1:200), and a mouse anti--tubulin antibody B-5-1-2 (Sigma, 1:1000) were used as primary antibodies, and peroxidase-conjugated goat anti-rabbit (Jackson ImmunoResearch, West Grove, PA) and donkey anti-mouse (Chemicon) antibodies were used as secondary antibodies. For the signal detection, SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) was used as a substrate of peroxidase for signal detection. The chemiluminescent signals were obtained with a LAS1000 image analyzer (Fuji Film, Tokyo, Japan) and measured with Image Gauge software (Fuji Film).
Primary culture and transfection
Mixed cerebellar cell cultures were prepared from rats at embryonic day 18 according to previously published methods (Furuya et al., 1998; Tabata et al., 2000), except for the culture medium. Briefly, prepared cells were plated at a final density of 4x106 cells/ml on poly-L-ornithine-coated coverslips in 24-well plates filled with 40 µl of seeding solution (DMEM-F12 containing 10% fetal calf serum). After incubation for 3 hours at 37°C, 400 µl of culture medium [DMEM-F12 containing B27 (final dilution, 1:100; Gibco, Rockville, MD), N2 supplement (1:100; Gibco), and 0.5 ng/ml tri-iodothyronine (Sigma-Aldrich)] was added to the well. Cells were treated either with 5 µM KN-93, 1 µM TTX, 10 µM nifedipine, or 30 µM CPCCOEt during the appropriate periods indicated in the Results section. The stathmin-Myc or SCG10-Myc expression plasmid (1 µg) or siRNA (final concentration 100 nM) was transfected using Lipofectamine 2000 (Gibco) immediately after the addition of the culture medium at 0 days in vitro (DIV-0). In the siRNA experiment, PCs in which Alexa Fluor-546 signal was detected within the soma (revealed by calbindin signal) in z-axis-stacked confocal micrographs, were selected and observed.
Immunocytochemistry
Primary cultured cerebellar cells were fixed in 10% formaldehyde neutral buffer (pH 7.0) at 4°C for 45 minutes. After washes with PBS, the cells were treated with PBST at RT for 3 minutes, followed by two washes with PBS for 10 minutes each. The cells were then treated with blocking buffer (2% BSA in PBS) at RT for 1 hour. To detect stathmin-Myc transfectants, a rabbit anti-calbindin antibody (1:1000) and mouse anti-Myc antibody (1:100) diluted in blocking buffer were incubated with the cells at 4°C overnight. For detection of stathmin phosphorylated on Ser16, a mouse anti-calbindin antibody (1:250) and rabbit anti-stathmin phospho-Ser16 antibody (1:100) were used as primary antibodies. For observation of stathmin or SCG10 distribution, a rabbit anti-stathmin antibody (Sigma-Aldrich, 1:100) or goat anti-SCG10 antibody (1:100) and a mouse anti-calbindin antibody (1:250) were used as primary antibodies. After three washes with PBS for 10 minutes each, the cells were incubated with FITC- and rhodamine-conjugated secondary antibodies at 4°C overnight. Cells were washed with PBS (containing DRAQ5 for nuclear staining) three times for 10 minutes, and then mounted with the ProLong Gold antifade reagent (Molecular Probes). The fluorescent signals were examined using an LSM 5 PASCAL laser-scanning confocal microscope.
Data analysis
All micrograph analyses were performed using Metamorph Software (Molecular Devices, Downingtown, PA). The quantitative measurements of stathmin mRNA in Fig. 1D and stathmin phosphorylation levels in Fig. 4 were defined by the average signal intensity in the PC soma. To measure the signal length of MAP2 and acetylated -tubulin in the molecular layer in Fig. 2, and the dendritic length of PCs in Fig. 3, maximum-intensity projection images were prepared. The signal lengths in each of the images were traced and calculated using the `Distance' function in the software. All statistical analyses were performed using StatView Software (Abacus Concepts, Berkeley, CA).
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