Masakazu Kataoka

Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the α1A subunit of the P/Q-type calcium channel

Synaptotagmins are synaptic vesicle proteins containing two calcium‐binding C2 domains which are involved in coupling calcium influx through voltage‐gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q‐type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co‐immunoprecipitated [125I]ω‐conotoxin MVIIC‐labelled calcium channels. Direct interactions were studied between in vitro‐translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the α1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780–969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium‐independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4°C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore‐forming subunit of the P/Q‐type channel may optimally locate the calcium‐binding sites that initiate exocytosis within a zone of voltage‐gated calcium entry.

Protein kinase C‐mediated translocation of secretory vesicles to plasma membrane and enhancement of neurotransmitter release from PC12 cells

In order to elucidate the molecular mechanism of phorbol ester‐induced potentiation of neurotransmitter release, changes in the subcellular distribution of secretory vesicles were studied in PC12 cells. Dopamine (DA) and acetylcholine containing vesicles were selectively labelled by expressing green fluorescent protein‐conjugated vesicular monoamine transporter and vesicular acetylcholine transporter, respectively. In the resting state, these vesicles were distributed throughout the cytoplasm. Phorbol‐12‐myristate‐13‐acetate (PMA), but not the inactive analogue 4α‐PMA, induced a redistribution of both types of secretory vesicles near the plasma membrane, and this change was abolished by a protein kinase C (PKC) inhibitor, bisindolylmaleimide I (BIS). PMA also induced a marked enhancement of depolarization‐induced DA release and phosphorylation of SNAP‐25 at Ser187. BIS completely inhibited PMA‐induced SNAP‐25 phosphorylation but suppressed PMA‐induced enhancement of DA release only partially. These results suggest that PMA enhances neurotransmitter release from PC12 cells by both PKC‐dependent and PKC‐independent mechanisms, and PKC enhances neurotransmitter release by recruiting secretory vesicles to the plasma membrane.

Nerve growth factor-induced phosphorylation of SNAP-25 in PC12 cells : a possible involvement in the regulation of SNAP-25 localization

Abstract: Synaptosomal‐associated protein of 25 kDa (SNAP‐25), a t‐SNARE protein essential for neurotransmitter release, is phosphorylated at Ser187 following activation of cellular protein kinase C by treatment with phorbol 12‐myristate 13‐acetate. However, it remains unclear whether neuronal activity or an endogenous ligand induces the phosphorylation of SNAP‐25. Here we studied the phosphorylation of SNAP‐25 in PC12 cells using a specific antibody for SNAP‐25 phosphorylated at Ser187. A small fraction of SNAP‐25 was phosphorylated when cells were grown in the absence of nerve growth factor (NGF). A brief treatment with NGF that was enough to activate the mitogen‐activated protein kinase signal transduction pathway did not increase the phosphorylation of SNAP‐25; however, phosphorylation was up‐regulated after a prolonged incubation with NGF. Up‐regulation was transitory, and maximum phosphorylation (a fourfold increase over basal phosphorylation) was achieved between 36 and 48 h after the addition of NGF. Immunofluorescent microscopy showed that SNAP‐25 was localized primarily in the plasma membrane, although a significant population was also present in the cytoplasm. Quantitative microfluorometry revealed that prolonged treatment with NGF resulted in a preferential localization of SNAP‐25 in the plasma membrane. A mutational study using a fusion protein with green fluorescent protein as a tag indicated that the point mutation of Ser187 to Ala abolished the NGF‐dependent relocalization. A population of SNAP‐25 in the plasma membrane was not increased by a point mutation at Ser187 to Glu; however, it was increased by prolonged treatment with NGF, indicating that the SNAP‐25 phosphorylation is essential, but not sufficient, for the NGF‐induced relocation to the plasma membrane. Our results suggest a close temporal relationship between the up‐regulation of SNAP‐25 phosphorylation and its relocation, and NGF‐induced differentiation of PC12 cells.

A Single Amino Acid Mutation in SNAP-25 Induces Anxiety-Related Behavior in Mouse

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser187 of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser187 of SNAP-25 with Ala using “knock-in” technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.

VAMP-2 promotes neurite elongation and SNAP-25A increases neurite sprouting in PC12 cells.

Recent studies suggest that the soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE)-mediated membrane fusion system is involved in vesicle fusion in the plasma membrane that allows expansion for neurite elongation. There have been several reports analyzing the effects of neurite outgrowth by inhibition of SNAREs. In this study, we took the opposite approach by overexpressing green fluorescent protein (GFP)-fusion SNAREs, including VAMP-2, SNAP-25A, and syntaxin1A, in PC12 cells to investigate the role of SNAREs in the neurite outgrowth of PC12 cells. Neurite outgrowth analysis demonstrated that: (1) GFP-VAMP-2 increased the length of individual neurites, without changing the number of neurites per cell; (2) GFP-SNAP-25A increased the number of neurites per cell, with no change in the length of the individual neurites. In both cases, the total length of neurites per cell was increased; (3) GFP-syntaxin1A resulted in no significant change, either in neurite length, or in the number of neurites per cell. These findings suggest that when overexpressed in PC12 cells, VAMP-2 can promote neurite elongation, while SNAP-25A can stimulate neurite sprouting. On the other hand, overexpression of syntaxin1A neither promotes nor inhibits neurite outgrowth. Thus VAMP-2 and SNAP-25A play different roles in neurite elongation and sprouting.

Accumulation of α-Synuclein Triggered by Presynaptic Dysfunction

Pathological examination of dementia with Lewy bodies patients identified the presence of abnormal α-synuclein (αSyn) aggregates in the presynaptic terminals. αSyn is involved in the regulation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Importantly, αSyn-transgenic mouse and postmortem examination of patients with Parkinsons disease have demonstrated the abnormal distribution of SNARE protein in presynaptic terminals. In this study, we investigated the effects of SNARE dysfunction on endogenous αSyn using Snap25S187A/S187A mutant mice. These mice have homozygous knock-in gene encoding unphosphorylatable S187A-substituted synaptosomal-associated protein of 25 kDa (SNAP-25). The mice displayed a significant age-dependent change in the distribution of αSyn and its Ser129-phosphorylated form in abnormally hypertrophied glutamatergic nerve terminals in the striatum. Electron-microscopic analysis revealed the abnormally condensed synaptic vesicles with concomitant mislocalization of αSyn protein to the periactive zone in the glutamatergic nerve terminals. However, the Snap25S187A/S187A mutant mouse harbored no abnormalities in the nigrostriatal dopaminergic neurons. Our present results suggest that SNARE dysfunction is the initial trigger of mislocalization and accumulation of αSyn, and probably is an important pathomechanism of α-synucleinopathies.

The TraB protein, which mediates the intermycelial transfer of the Streptomyces plasmid pSN22, has functional NTP‐binding motifs and is localized to the cytoplasmic membrane

The traB gene on the Streptomyces conjugative plasmid pSN22 is required for intermycelial plasmid transfer and the mobilization of chromosomal markers (Cma). The predicted amino acid sequence of TraB contains one Walker type‐A and two type‐B NTP‐binding motifs. Site‐directed mutagenesis revealed that the type‐A motif and one of the type‐B motifs, 109 amino acid residues downstream of the type‐A motif, were essential for both plasmid transfer and Cma. The second type‐B sequence could be changed without any phenotypic effect. A modified traB gene was constructed, resulting in the production of a functional protein with an amino‐terminal c‐Myc epitope tag for immunological analysis. This protein was associated with the cytoplasmic membrane, suggesting that TraB is a membrane protein that uses energy from ATP hydrolysis to transport DNA between mycelia. The c‐Myc tagging of TraB decreased the efficiency of intramycelial plasmid spread, suggesting that TraB is involved in both inter‐ and intramycelial transfer processes.

Raman and Fluorescence Spectroscopic Studies of a DNA-Dispersed Double-Walled Carbon Nanotube Solution

We performed resonant Raman/fluorescence spectroscopic studies on double-walled carbon nanotubes (DWNTs) that were dispersed in an aqueous single stranded DNA solution. The luminescence signals from the inner tubes of DWNTs are intensified in the isolated state of each individual DWNT. The completely depressed radial breathing modes (RBMs) associated with the outer tubes (whether semiconducting or metallic) via the mechanical wrapping and the strong charge transfer between DNA and the outer tubes support our interpretation that the bright luminescence and sharp absorption spectra come from only the inner tubes, and not from isolated SWNTs. The circumferentially wrapped DNA on the outer tubes of individually isolated DWNTs in an aqueous solution gives rise to strong charge transfer to the semiconducting and metallic outer tubes as well as to generating physical strain in the outer tubes.

Development- and activity-dependent regulation of SNAP-25 phosphorylation in rat brain

Synaptosomal-associated protein of 25kDa (SNAP-25), a member of the SNARE proteins essential for neurotransmitter release, is phosphorylated at Ser(187) in PC12 cells and in the rat brain in a protein kinase C-dependent manner. It remains unclear how the phosphorylation of SNAP-25 is regulated during development and by neuronal activity. We studied the mode of SNAP-25 phosphorylation at Ser(187) in the rat brain using an anti-phosphorylated SNAP-25 antibody. Both the expression and phosphorylation of SNAP-25 increased remarkably during the early postnatal period, but their onsets were quite different. SNAP-25 expression was detected as early as embryonic Day 18, whereas the phosphorylation of SNAP-25 could not be detected until postnatal Day 4. A delay in the onset of phosphorylation was also observed in cultured rat hippocampal neurons. The phosphorylation of SNAP-25 was regulated in a neuronal activity-dependent manner and, in the rat hippocampus, decreased by introducing seizures with kainic acid. These results clearly indicated that the phosphorylation of SNAP-25 at Ser(187) is regulated in development- and neuronal activity-dependent manners, and is likely to play important roles in higher brain functions.

Defect‐Enhanced Dispersion of Carbon Nanotubes in DNA Solutions

Since DNA, a well-known biopolymer, has proven to be effective for dispersing and sorting carbon nanotubes, intensive studies have been carried out in order to obtain both theoretical and experimental understanding of the DNA–nanotube interaction. The ultrasonication process has been applied to dispersing a strongly bundled aggregate of carbon nanotubes in a nanotube suspension containing DNA in order to overcome the attractive van der Waals forces between adjacent tubes. The strong hydrodynamic shear forces generated by the ultrasonication process create a space within the bundled nanotubes, which allows the DNA molecules to impinge and become adsorbed on the outer surface of the nanotubes through a hydrophobic interaction, thereby leading to a stable nanotube emulsion through the formation of DNA– nanotube hybrid structures. Until now, single-stranded DNA has proven to be more effective for dispersing nanotubes than double-stranded DNA, and the combination of the short complementary oligonucleotides d(GT)3 and d(AC)3 exhibit an even superior dispersing ability. Moreover, theoretical studies have assumed that the shape of carbon nanotubes constitutes a perfect cylinder. However, the catalytic growth of carbon nanotubes in the reaction chamber inevitably involves the formation of defects (e.g. vacancies, nonhexagonal rings, and foreign atoms), which modify the electronic structure and surface properties of the tubes 14] and are expected to ultimately alter the interaction between nanotubes and DNA. In order to be able to reproducibly assemble nanotube–DNA hybrid structures as bio-medical devices, it is essential to consider the effects of defects within carbon nanotubes. Herein, the role of defects generated on the sidewall of carbon nanotubes is studied by directly comparing the dispersibility of defective and crystalline thin (small outer diameter) multi-walled carbon nanotubes (MWNTs) in aqueous DNA solutions. The dispersibility of nanotubes depend strongly on the density of the defects on the sidewall. More specifically, crystalline tubes thermally treated at 2300 8C exhibit a dispersibility twice as low as that of the as-grown tubes in aqueous DNA solutions. Theoretical calculations shown herein support the conclusion that defect sites exhibit high reactivity toward the adsorption of DNA. There are three reasons for selecting thin MWNTs rather than single-walled carbon nanotubes (SWNTs) for this study: 1) Thin MWNTs exhibit a strongly bundled structure like SWNTs because of their small diameter below 10 nm, 2) they are structurally stable up to 2800 8C, while SWNTs and DWNTs are easily transformed into graphitic material at these high temperatures, 17] and 3) their diameter is too large (above 2 nm) for the manifestation of detailed quantum effects associated with 1D dispersion relations. As a general metric for comparing the structural integrity of SWNTs or DWNTs, the intensity of the Dband, which is explained by double-resonance theory, 19] is suitable for evaluating the quality of SWNTs containing only a small number of defects. However, due to the breakdown of the van Hove singularities, this approach is not applicable to SWNTs or MWNTs containing many defects. As a tool for controlling the defect density in as-grown defective thin MWNTs, we choose high-temperature thermal treatment 21] instead of the commonly used chemical treatment. Oxidative chemical treatment can introduce both chemical moieties and defects on the sidewall of the tubes, which might affect the tube–tube interactions as well as their dispersibility. In other words, it is difficult to separate the role of defects within tubes from the effects of chemical treatment in evaluating the dispersibility of tubes in DNA solutions. The effects of high-temperature annealing on the defect density of carbon nanotubes are clearly observed as consecutive changes in the Raman spectra (Figure 1 a). Although there are no distinct changes in the G-band located at 1582 cm 1 (E2g2 graphite mode), the intensities of the D-band (a defect-induced mode) at 1350 cm 1 decrease with increasing thermal treatment temperatures, and saturate for samples prepared at 2300 8C in argon. This temperature range is consistent with the region in conventional carbon materials where the mobility of carbon atoms increases abruptly. Therefore, a drastic decrease in the R value (the intensity of the D-band divided by the intensity of the G-band) from 0.42 to 0.1 (see inset in Figure 1 a) indicates the effective removal (or annealing) of defects within carbon nanotubes by the high-temperature thermal treatment. In order to visualize the improvement of the structural integrity, we carried out detailed SEM and TEM studies on pristine tubes (Figures 1 b, c) and tubes thermally treated at 2300 8C (FigACHTUNGTRENNUNGures 1 d, e). Pristine tubes consisting of undulated fringes and amorphous carbon layers (Figure 1 c) were transformed into crystalline tubes (Figure 1 e) featuring straight linear fringes lengthwise along the tube. Fortunately, there were no appa[a] J. H. Kim, Prof. M. Kataoka, Dr. D. Shimamoto, Prof. H. Muramatsu, Dr. Y. C. Jung, T. Tojo, Prof. T. Hayashi, Prof. Y. A. Kim, Prof. M. Endo Faculty of Engineering Shinshu University, 4-17-1 Wakasato, Nagano-shi 380-8553 (Japan) Fax: (+ 81) 26-269-5208 E-mail : yak@endomoribu.shinshu-u.ac.jp [b] Prof. M. Terrones Advanced Materials Department IPICYT, San Luis Potosi 78210 (M xico) [c] Prof. M. S. Dresselhaus Massachusetts Institute of Technology Cambridge, Massachusetts 02139-4307 (USA)