Doc2 has one Munc13-interacting domain at the N-terminal region and two C2-like domains interacting with Ca2+ and phospholipid at the C-terminal region. Doc2 consists of two isoforms, Doc2alpha and ...-beta. Doc2alpha is specifically expressed in neuronal cells and implicated in Ca2+-dependent neurotransmitter release, whereas Doc2beta is ubiquitously expressed and its function is unknown. We show here that both Doc2alpha and -beta interact with rat tctex-1, a light chain of cytoplasmic dynein, in both cell-free and intact cell systems. Overexpression of the N-terminal fragment of Doc2 containing the tctex-1-interacting domain induces changes in the intracellular localization of cation-independent mannose 6-phosphate receptor and its ligand, cathepsin D, which are transported from trans-Golgi network to late endosomes. Overexpression of the C-terminal fragment containing two C2-like domains shows the similar effect, but to a lesser extent, whereas overexpression of full-length Doc2 or the C-terminal fragment of rabphilin3 containing two C2-like domains does not show this effect. Because dynein is a minus-end-directed microtubule-based motor protein, these results suggest that Doc2, especially Doc2beta, plays a role in dynein-dependent intracellular vesicle transport.
We previously isolated a new protein having two C2-like domains which interacted with Ca2+ and phospholipid and named Doc2 (Double C2). Because Doc2 was abundantly expressed in brain where it was ...highly concentrated on the synaptic vesicle fraction, we have examined here whether Doc2 is involved in Ca2+-dependent exocytosis from cultured PC12 cells. For this purpose, we took advantage of the growth hormone (GH) co-expression assay system of PC12 cells in which GH is stored in dense core vesicles and released in response to high K+ in an extracellular Ca2+-dependent manner. Northern and Western blot analyses indicated that Doc2 is present in PC12 cells. Overexpression of hemagglutinin-tagged Doc2 stimulated the Ca2+-dependent, high K+-induced release of co-expressed GH without affecting the basal release. In the PC12 cells transfected with a plasmid with the coding sequence of Doc2 in the antisense orientation, the high K+-induced release of co-expressed GH was inversely inhibited. The Doc2 mutant expressing an N-terminal fragment or a C-terminal fragment containing two C2-like domains inhibited the high K+-induced release of co-expressed GH. These results indicate that Doc2 enhances Ca2+-dependent exocytosis of dense core vesicles from PC12 cells.
Munc13 is a component of the neurotransmitter release machinery which is specifically expressed in brain. Munc13 interacts with Doc2 and syntaxin which are also implicated in the neurotransmitter ...release process. Here we isolated another Munc13-interacting molecule from a rat brain cDNA library by use of the yeast two-hybrid system, identified it to be a novel type of β spectrin, and named it βSpIIIΣ1. βSpIIIΣ1 was specifically expressed in brain, where it was enriched in the synaptic vesicle and plasma membrane fractions. Because spectrin has been shown to interact with the actin cytoskeleton which is involved in the exocytotic process, the present results suggest that the Munc13-βSpIIIΣ1 interactions play a role in neurotransmitter release.
We have previously isolated from a human brain cDNA library, a new protein having two C2-like domains which interact with Ca
2+ and phospholipid, and named Doc2α. Doc2α is abundantly expressed in ...brain, where it is highly concentrated on the synaptic vesicle fraction, and is implicated in Ca
2+-dependent exocytosis. We have isolated here a mouse Doc2α cDNA and determined the localization of its mRNA in adult mouse brain. The amino acid sequence of the mouse Doc2α cDNA is 92% identical with that of the human counterpart. Northern blot analysis and in situ hybridization on adult mouse brain sections have revealed that Doc2α is predominantly expressed in mouse brain, where it is expressed in neuronal cells, but not in non-neuronal cells. Doc2α is highly expressed in the olfactory bulb, cerebral cortex, hippocampus, amygdaloid complex, and ventromedial hypothalamus nucleus, but not in the cerebellum, caudate-putamen, or ventral thalamus. These results indicate that Doc2α is expressed heterogeneously in mouse brain, where it is predominantly expressed in neuronal cells, and suggest that Doc2α plays a specific role in the area where it is expressed.
Cerebrospinal fluid prostaglandin E(2) (PGE(2)) levels are elevated in patients with Alzheimer's disease (AD), suggesting an involvement of PGE(2) in the neurodegeneration. AD is characterized by ...deposits of amyloid beta protein (Abeta) in various regions of the brain, e.g. the cerebral cortex. In the present study, we investigated the effects of PGE(2) on neuronal survival in primary cultures of rat cortical neurons. PGE(2) had no effect on neuronal cell viability or its morphology. Therefore, we examined the synergistic effects of PGE(2) with Abeta, a neurotoxin. Abeta caused neuronal cell death via apoptosis. PGE(2) significantly suppressed Abeta neurotoxicity, but did not promote the neurotoxicity. Furthermore, PGE(2) ameliorated Abeta-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Abeta increased the influx of Ca(2+) into neurons before cell death. Nimodipine, an inhibitor of the L-type voltage-sensitive calcium channel (L-VSCC), significantly reduced Abeta-potentiated Ca(2+) uptake. On the other hand, there was no effect on the Abeta-induced Ca(2+) influx by an N-VSCC blocker or P/Q-VSCC blockers. Moreover, the inhibitor of L-VSCC suppressed Abeta-induced neuronal cell death, whereas neither an N-VSCC blocker nor P/Q-VSCC blockers affected the neurotoxicity of Abeta. PGE(2) also suppressed the Abeta-induced Ca(2+) influx in a concentration-dependent manner. This study demonstrated that PGE(2) rescues cortical neurons from Abeta-induced apoptosis by reducing Ca(2+) influx in the primary culture. Furthermore, the present study suggested that the inhibition of L-VSCC contributes to the neuroprotective effect of PGE(2).
Previously we demonstrated the gene transduction of granulocyte- colony stimulating-factor (GM-CSF) gene into murine monocytic leukemia cell line of WEHI3B eliminated the tumorigenesity in vivo.The ...rejection process of subcutaneous tumor was as follows; transient tumor growth peaked around 10 day after tumor injection, then the tumors were rejected within a week. We analyzed the gene expression of the transiently established tumor masses by the serial analysis of gene expression (SAGE) method to identify molecules associated with the antitumor effect. We identified that chemokine genes of TARC and RANTES were preferentially expressed in the GM-CSF-transduced tumors and the contribution of the two genes to the regression of GM-CSF-transduced tumors was suggested. To confirm the finding, we observed in vivo tumor formation of WEHI-3B cells transduced with a retroviral vector expressing murine GM- CSF, TARC, RANTES, GM-CSF+TARC or GM-CSF+RANTES, respectively. 1.0x10^6^cells of each transduced cell group and non- transduced cell group were inoculated subcutaneously into syngeneic mice, female BALB/c (n=20 or 21). Then tumorigenecity and survival rate were observed. Both of the combination of GM-CSF with TARC or RANTES gene transduction profoundly inhibited tumor formation. In addition, the 2 groups demonstrated significantly longer survival than GM-CSF transduced group. Immunohistochemistry of the tumor section at day 10 after inoculation revealed more significant contribution of CD4+ T cells to tumor regression in both of the TARC/GM-CSF and RANTES/ GM-CSF-transduced cell tumors than GM-CSF transduced cell tumors. More CD11c positive cells were observed in RANTES/GM-CSF-transduced cell tumors than GM-CSF transduced cell tumors. To determine the therapeutic effects of tumor vaccines producing GM-CSF and TARC and/or RANTES against established tumor, in vivo mouse experiments are under way.
Doc2α is a synaptic vesicle‐associated Ca2 + ‐binding protein. To study the role of Doc2α in synaptic transmission and modulation, we generated homozygous null Doc2α mutant mice. In the CA1 region of ...hippocampal slices in the mutant mice, excitatory synaptic responses evoked with prolonged 5 Hz stimulation showed a significantly larger frequency facilitation followed by a steeper depression than those in wild‐type mice, whereas there was no difference in synaptic transmission at lower frequencies or in paired‐pulse facilitation. These results suggest that Doc2α regulates synaptic transmission when high Ca2 + concentrations in the presynaptic terminal are sustained. Furthermore, the mutant mice showed impairment in long‐term potentiation and passive avoidance task. Thus, Doc2α may regulate transmitter release during repetitive synaptic activation, thereby contributing to memory formation.
Abstract
Accumulation of amyloid beta peptide (Abeta) in brain is a hallmark of Alzheimer's disease (AD). Inhibition of beta‐site amyloid precursor protein (APP)‐cleaving enzyme‐1 (BACE1), the enzyme ...that initiates Abeta production, and other Abeta‐lowering strategies are commonly tested in transgenic mice overexpressing mutant APP. However, sporadic AD cases, which represent the majority of AD patients, are free from the mutation and do not necessarily have overproduction of APP. In addition, the commonly used Swedish mutant APP alters APP cleavage. Therefore, testing Abeta‐lowering strategies in transgenic mice may not be optimal. In this study, we investigated the impact of BACE1 inhibition in non‐transgenic mice with physiologically relevant APP expression. Existing Abeta ELISAs are either relatively insensitive to mouse Abeta or not specific to full‐length Abeta. A newly developed ELISA detected a significant reduction of full‐length soluble Abeta 1–40 in mice with the BACE1 homozygous gene deletion or BACE1 inhibitor treatment, while the level of x‐40 Abeta was moderately reduced due to detection of non‐full‐length Abeta and compensatory activation of alpha‐secretase. These results confirmed the feasibility of Abeta reduction through BACE1 inhibition under physiological conditions. Studies using our new ELISA in non‐transgenic mice provide more accurate evaluation of Abeta‐reducing strategies than was previously feasible.
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