Keiichi Ueda

Amyloid β Protein Potentiates Ca2+ Influx Through L-Type Voltage-Sensitive Ca2+ Channels: A Possible Involvement of Free Radicals

Abstract: Amyloid β protein (Aβ), the central constituent of senile plaques in Alzheimers disease (AD) brain, is known to exert toxic effects on cultured neurons. The role of the voltage‐sensitive Ca2+ channel (VSCC) in β(25–35) neurotoxicity was examined using rat cultured cortical and hippocampal neurons. When L‐type VSCCs were blocked by application of nimodipine, β(25–35) neurotoxicity was attenuated, whereas application of ω‐conotoxin GVIA (ω‐CgTX‐GVIA) or ω‐agatoxin IVA (ω‐Aga‐IVA), the blocker for N‐ or P/Q‐type VSCCs, had no effects. Whole‐cell patch‐clamp studies indicated that the Ca2+ current density of β(25–35)‐treated neurons is about twofold higher than that of control neurons. Also, β(25–35) increased Ca2+ uptake, which was sensitive to nimodipine. The 2′,7′‐dichlorofluorescin diacetate assay showed the ability of β(25–35) to produce reactive oxygen species. Nimodipine had no effect on the level of free radicals. In contrast, vitamin E, a radical scavenger, reduced the level of free radicals, neurotoxicity, and Ca2+ uptake. These results suggest that β(25–35) generates free radicals, which in turn, increase Ca2+ influx via the L‐type VSCC, thereby inducing neurotoxicity.

Amyloid β protein-induced neuronal cell death: neurotoxic properties of aggregated amyloid β protein

The neurotoxic effects of soluble and aggregated synthetic amyloid beta protein (A beta P) have been investigated in rat primary cultures. Freshly solubilized beta(1-40) was neurotoxic not to immature, but to mature hippocampal neurons. On the other hand, aggregated beta(1-40) was neurotoxic to both. Neurotoxicity induced by aggregated beta(1-40) was 10-fold more potent than soluble beta(1-40) and was not prevented by substance P. The neurotoxicity of aggregated beta(1-40) to cultured neurons depended on the peptide concentration and the duration of exposure to it. Cerebral cortical and hippocampal neurons were significantly susceptible to aggregated beta(1-40) than cerebellar granular cells, and cultured astrocytes were not vulnerable to aggregated beta(1-40) even at high concentrations.

Group IB secretory phospholipase A2 induces neuronal cell death via apoptosis

Group IB secretory phospholipase A2 (sPLA2‐IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high‐affinity binding sites of sPLA2‐IB have been documented. However, it remains obscure whether sPLA2‐IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2‐IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2‐IB induced neuronal cell death in a concentration‐dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2‐IB‐induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2‐IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Δ12‐PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase‐2 (COX‐2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2‐IB‐induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2‐IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Δ12‐PGJ2, might mediate sPLA2‐IB‐induced apoptosis.

Gas6 rescues cortical neurons from amyloid β protein-induced apoptosis

Abstract Gas6, a product of the growth-arrest-specific gene 6, protects neurons from serum deprivation-induced apoptosis. Neuronal apoptosis is also caused by amyloid β protein (Aβ), whose accumulation in the brain is a characteristic feature of Alzheimer’s disease. Aβ induces Ca2+ influx via L-type voltage-dependent calcium channels (L-VSCCs), leading to its neurotoxicity. In the present study, we investigated effects of Gas6 on Aβ-induced cell death in primary cultures of rat cortical neurons. Aβ caused neuronal cell death in a concentration- and time-dependent manner. Gas6 significantly prevented neurons from Aβ-induced cell death. Gas6 ameliorated Aβ-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Prior to cell death, Aβ increased influx of Ca2+ into neurons through L-VSCCs. Gas6 significantly inhibited the Aβ-induced Ca2+ influx. The inhibitor of L-VSCCs also suppressed Aβ-induced neuronal cell death. The present 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 Aβ-induced apoptosis. Furthermore, the present study indicates that inhibition of L-VSCC contributes to the neuroprotective effect of Gas6.

Human group IIA secretory phospholipase A2 potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels in cultured rat cortical neurons

Mammalian group IIA secretory phospholipase A2 (sPLA2‐IIA) generates prostaglandin D2 (PGD2) and triggers apoptosis in cortical neurons. However, mechanisms of PGD2 generation and apoptosis have not yet been established. Therefore, we examined how second messengers are involved in the sPLA2‐IIA‐induced neuronal apoptosis in primary cultures of rat cortical neurons. sPLA2‐IIA potentiated a marked influx of Ca2+ into neurons before apoptosis. A calcium chelator and a blocker of the L‐type voltage‐sensitive Ca2+ channel (L‐VSCC) prevented neurons from sPLA2‐IIA‐induced neuronal cell death in a concentration‐dependent manner. Furthermore, the L‐VSCC blocker ameliorated sPLA2‐IIA‐induced morphologic alterations and apoptotic features such as condensed chromatin and fragmented DNA. Other blockers of VSCCs such as N type and P/Q types did not affect the neurotoxicity of sPLA2‐IIA. Blockers of L‐VSCC significantly suppressed sPLA2‐IIA‐enhanced Ca2+ influx into neurons. Moreover, reactive oxygen species (ROS) were generated prior to apoptosis. Radical scavengers reduced not only ROS generation, but also the sPLA2‐IIA‐induced Ca2+ influx and apoptosis. In conclusion, we demonstrated that sPLA2‐IIA potentiates the influx of Ca2+ into neurons via L‐VSCC. Furthermore, the present study suggested that eicosanoids and ROS generated during arachidonic acid oxidative metabolism are involved in sPLA2‐IIA‐induced apoptosis in cooperation with Ca2+.

Effects of endothelin B receptor agonists on amyloid β protein (25-35)-induced neuronal cell death

Endothelin (ET), a vasoconstrictive peptide, acts as an anti-apoptotic factor, and endothelin receptor B (ET(B) receptor) is associated with neuronal survival in the brain. In the Alzheimers disease (AD) brain, accumulation of amyloid beta protein (Abeta) is thought to cause neuronal cell death via apoptosis. In the present study, we investigated effects of ET(B) receptor agonists on Abeta-induced neuronal cell death. In primary cultures of rat cortical neurons, Abeta(25-35) caused neuronal cell death in a concentration- and time-dependent manner. Abeta(25-35)-induced neuronal cell death was accompanied by chromatin condensation and DNA fragmentation, exhibiting apoptotic features. ET-3 and IRL-1620, ET(B) receptor agonists, significantly prevented neurons from undergoing Abeta(25-35)-induced cell death. Prior to cell death, Abeta increased concentration of intracellular Ca(2+) ([Ca(2+)](i)). Nimodipine, an L-type voltage-sensitive Ca(2+) channel (L-VSCC) blocker, suppressed the Abeta-induced Ca(2) influx, and attenuated Abeta-induced neuronal apoptosis. On the other hand, omega-conotoxin GIVA, an N-type VSCC blocker and omega-conotoxin MVIIC and omega-agatoxin IVA, P/Q-type VSCC blockers, had no effect. ET-3 and IRL-1620 significantly blocked Abeta(25-35)-induced Ca(2) influx. Furthermore, BQ788, an ET(B) receptor antagonist, inhibited both an anti-apoptotic effect and an L-VSCC-inactivating effect of ET(B) receptor agonists. In conclusion, ET(B) receptor agonists exhibit a protective effect against neurotoxicity of Abeta. Furthermore, these agonists appear to act as anti-apoptotic factors by blocking of L-VSCCs.

Effects of S-2474, a novel nonsteroidal anti-inflammatory drug, on amyloid β protein-induced neuronal cell death

The accumulation of amyloid β protein (Aβ) in the brain is a characteristic feature of Alzheimers disease (AD). Clinical trials of AD patients with nonsteroidal anti‐inflammatory drugs (NSAIDs) indicate a clinical benefit. NSAIDs are presumed to act by suppressing inhibiting chronic inflammation in the brain of AD patients. In the present study, we investigated effects of S‐2474 on Aβ‐induced cell death in primary cultures of rat cortical neurons. S‐2474 is a novel NSAID, which inhibits cyclo‐oxygenase‐2 (COX‐2) and contains the di‐tert‐butylphenol antioxidant moiety. S‐2474 significantly prevented neurons from Aβ(25 – 35)‐ and Aβ(1 – 40)‐induced cell death. S‐2474 ameliorated Aβ‐induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA completely. Prior to cell death, Aβ(25 – 35) generated prostaglandin D2 (PGD2) and free radicals from neurons. PGD2 is a product of cyclo‐oxygenase (COX), and caused neuronal cell death. S‐2474 significantly inhibited the Aβ(25 – 35)‐induced generation of PGD2 and free radicals. The present cortical cultures contained little non‐neuronal cells, indicating that S‐2474 affected neuronal survival directly, but not indirectly via non‐neuronal cells. Both an inhibitory effect of COX‐2 and an antioxidant effect might contribute to the neuroprotective effects of S‐2474. In conclusion, S‐2474 exhibits protective effects against neurotoxicity of Aβ. Furthermore, the present study suggests that S‐2474 may possess therapeutic potential for AD via ameliorating degeneration in neurons as well as suppressing chronic inflammation in non‐neuronal cells.

Protein kinase inhibitor attenuates apoptotic cell death induced by amyloid β protein in culture of the rat cerebral cortex

Amyloid beta protein (A beta) is deposited characteristically in the brain of patients with Alzheimers disease. Effects of protein kinase inhibitors (H-89, H-7, KN-62) on A beta-induced neuronal cell death were examined in primary culture of dissociated cerebral cortical cells. beta(25-35), the active fragment of A beta, induced neuronal cell death with apoptotic features including chromatin condensation and DNA fragmentation. The cell death was attenuated by cycloheximide or by H-89, a specific protein kinase A (PKA) inhibitor, but not by H-7 or KN-62. These data suggest that beta(25-35) induces apoptotic cell death through the PKA-mediated pathway.

Prostaglandin E2 rescues cortical neurons from amyloid β protein-induced apoptosis

Cerebrospinal fluid prostaglandin E(2) (PGE(2)) levels are elevated in patients with Alzheimers 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).

Porcine pancreatic group IB secretory phospholipase A2potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels

Secretory phospholipase A(2) (sPLA(2)) exhibits neurotoxicity in the central nervous system. There are high-affinity binding sites of the porcine pancreatic group IB sPLA(2) (sPLA(2)-IB) in the brain. sPLA(2)-IB causes neuronal cell death via apoptosis in the rat cerebral cortex. Although apoptosis is triggered by an influx of Ca(2+) into neurons, it has not yet been ascertained whether the Ca(2+) influx is associated with the neurotoxicity of sPLA(2)-IB. We thus examined the possible involvement of Ca(2+) in the neurotoxicity of sPLA(2)-IB in the primary culture of rat cortical neurons. sPLA(2)-IB induced neuronal cell death in a concentration- and time-dependent manner. This death was accompanied by condensed chromatin and fragmented DNA, exhibiting apoptotic features. Before apoptosis, sPLA(2)-IB markedly enhanced the influx of Ca(2+) into neurons. A calcium chelator suppressed neurons from sPLA(2)-IB-induced neuronal cell death in a concentration-dependent manner. An L-type voltage-sensitive Ca(2+) channel (L-VSCC) blocker significantly protected the sPLA(2)-IB-potentiated influx of Ca(2+). On the other hand, blockers of N-VSCC and P/Q-VSCC did not. An L-VSCC blocker protected neurons from sPLA(2)-IB-induced neuronal cell death. In addition, the L-VSCC blocker ameliorated the apoptotic features of sPLA(2)-IB-treated neurons. Neither an N-VSCC blocker nor P/Q-VSCC blockers affected the neurotoxicity of the enzyme. In conclusion, these findings demonstrate that the influx of Ca(2+) into neurons play an important role in the neurotoxicity of sPLA(2)-IB. Furthermore, the present study suggests that L-VSCC contribute to the sPLA(2)-IB-potentiated influx of Ca(2+) into neurons.