Alzheimer’s disease (AD) is a neurodegenerative affliction associated with memory dysfunction. Senile plaques are a pathological hallmark of AD, and amyloid β (Aβ) peptides are a major component of ...these plaques. Aβ peptides are derived from proteolytic cleavage of the Aβ protein precursor (APP) by β- and γ-secretases to generate two principal species, Aβ1–40 and Aβ1–42. We have developed antibodies against the N- and C-termini of these peptides, and an ELISA for accurate and sensitive quantitative assessment. Sandwich ELISA composed of N-terminus (Aβ1) end-specific antibody, clone 82E1, and C-termini end-specific antibodies, and clones 1A10 and 1C3 for Aβ40 and Aβ42, respectively, detects full-length Aβ1–40 and 1–42 with a sensitivity in the sub single digit fmol/ml (equivalent to single digit pg/ml) range with no cross-reactivity to APP. A combination of C-termini antibodies and an antibody against the middle region of Aβ detects mouse Aβ in non-transgenic mouse brains.
Amyloid β protein (Aβ)- and human group IIA secretory phospholipase A
2 (sPLA
2-IIA)-induced neuronal cell death have been established as
in vitro models for Alzheimer’s disease (AD) and stroke. Both ...sPLA
2-IIA and Aβ causes neuronal apoptosis by increasing the influx of Ca
2+ through L-type voltage-sensitive Ca
2+ channel (L-VSCC). In the present study, we evaluated effects of a selective L-VSCC blocker,
S-(+)-methyl 4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitro-phenyl)thieno2,3-
bpyridine-5-carboxylate (
S-312-
d), on Aβ- and sPLA
2-IIA-induced neuronal apoptosis in primary cultures of rat cortical neurons.
S-312-
d significantly rescued cortical neurons from Aβ- and sPLA
2-IIA-induced cell death. Both cell death stimuli caused the appearance of apoptotic features such as plasma membrane blebs, chromatin condensation, and DNA fragmentation.
S-312-
d completely suppressed these apoptotic features. Before apoptosis, the two death ligands markedly enhanced an influx of Ca
2+ into neurons.
S-312-
d significantly prevented neurons from sPLA
2-IIA- and Aβ-induced Ca
2+ influx. Furthermore, the neuroprotective effect of
S-312-
d was more potent than that of another L-VSCC blocker, nimodipine. On the other hand, blockers of other VSCCs such as the N-type and P/Q-type calcium channels had no effect on the neuronal cell death, apoptotic features and Ca
2+ influx. In conclusion, we demonstrated that
S-312-
d rescues cortical neurons from Aβ- and sPLA
2-IIA-induced apoptosis.
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, Doc2α and -β. ...Doc2α is specifically expressed in neuronal cells and implicated in Ca2+-dependent neurotransmitter release, whereas Doc2β is ubiquitously expressed and its function is unknown. We show here that both Doc2α and -β 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 Doc2β, plays a role in dynein-dependent intracellular vesicle transport.
The proinflammatory cytokine interleukin (IL)-1beta is up-regulated in microglial cells surrounding amyloid plaques, leading to the hypothesis that IL-1beta is a risk factor for Alzheimer's disease. ...However, we unexpectedly found that IL-1beta significantly enhanced alpha-cleavage, indicated by increases in sAPPalpha and C83, but reduced beta-cleavage, indicated by decreases in sAPPbeta and Abeta40/42, in human neuroblastoma SK-N-SH cells. IL-1beta did not significantly alter the mRNA levels of BACE1, ADAM-9, and ADAM-10, but up-regulated that of TACE by threefold. The proform and mature form of TACE protein were also significantly up-regulated. A TACE inhibitor (TAPI-2) concomitantly reversed the IL-1beta-dependent increase in sAPPalpha and decrease in sAPPbeta, suggesting that APP consumption in the alpha-cleavage pathway reduced its consumption in the beta-cleavage pathway. IL-1Ra, a physiological antagonist for the IL-1 receptor, reversed the effects of IL-1beta, suggesting that the IL-1beta-dependent up-regulation of alpha-cleavage is mediated by the IL-1 receptor. IL-1beta also induced this concomitant increase in alpha-cleavage and decrease in beta-cleavage in mouse primary cultured neurons. Taken together we conclude that IL-1beta is an anti-amyloidogenic factor, and that enhancement of its signaling or inhibition of IL-1Ra activity could represent potential therapeutic strategies against Alzheimer's disease. PUBLICATION ABSTRACT
Accumulation of amyloid beta in the brain is a pathological hallmark of Alzheimer's disease, and the reduction of amyloid beta has been proposed as a primary therapeutic target. Mice immunized ...against amyloid beta and mice infused with anti-amyloid beta antibody (active and passive immunization, respectively) have reduced brain amyloid beta levels, and two mechanisms have been proposed: microglial phagocytosis in the brain and enhancement of amyloid beta efflux by antibodies present in the periphery (sequestration). The optimal antibody for microglial phagocytosis has been shown to be N-terminal-specific antibody; however, the potency of C-terminal-specific antibody in sequestration remains unclear. In this study, we found that anti-amyloid beta 40-specific antibody induces amyloid beta sequestration. These results indicate that C-terminal antibodies may be useful in amyloid beta sequestration therapy.
Gas6, a product of the growth-arrest-specific gene 6, protects cortical neurons from amyloid beta protein (Abeta)-induced apoptosis. Neuronal apoptosis is also caused by human group IIA secretory ...phospholipase A(2) (sPLA(2)-IIA), which is expressed in the cerebral cortex after brain ischemia. sPLA(2)-IIA induces Ca(2+) influx via L-type voltage-sensitive calcium channels (L-VSCCs), leading to its neurotoxicity. In the present study, we investigated effects of Gas6 on sPLA(2)-IIA-induced cell death in primary cultures of rat cortical neurons. sPLA(2)-IIA caused neuronal cell death in a concentration- and time-dependent manner. Gas6 significantly prevented neurons from sPLA(2)-IIA-induced cell death. Gas6 suppressed sPLA(2)-IIA-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Prior to cell death, sPLA(2)-IIA increased the influx of Ca(2+) into neurons through L-VSCCs. Gas6 significantly inhibited the sPLA(2)-IIA-induced Ca(2+) influx. The blocker of L-VSCCs also suppressed sPLA(2)-IIA-induced neuronal cell death. The cortical cultures contained few non-neuronal cells, indicating that Gas6 affected the survival of neurons directly, but not indirectly via non-neuronal cells. In conclusion, we demonstrate that Gas6 rescues cortical neurons from sPLA(2)-IIA-induced apoptosis. Furthermore, the present study indicates that inhibition of L-VSCC contributes to the neuroprotective effect of Gas6.
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