Gaku Sakaguchi

BACE1 inhibition reduces endogenous Abeta and alters APP processing in wild-type mice.

Accumulation of amyloid beta peptide (Abeta) in brain is a hallmark of Alzheimers 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.

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.

Interleukin-1β up-regulates TACE to enhance α-cleavage of APP in neurons: resulting decrease in Aβ production

The proinflammatory cytokine interleukin (IL)‐1β is up‐regulated in microglial cells surrounding amyloid plaques, leading to the hypothesis that IL‐1β is a risk factor for Alzheimer’s disease. However, we unexpectedly found that IL‐1β significantly enhanced α‐cleavage, indicated by increases in sAPPα and C83, but reduced β‐cleavage, indicated by decreases in sAPPβ and Aβ40/42, in human neuroblastoma SK‐N‐SH cells. IL‐1β 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‐1β‐dependent increase in sAPPα and decrease in sAPPβ, suggesting that APP consumption in the α‐cleavage pathway reduced its consumption in the β‐cleavage pathway. IL‐1Ra, a physiological antagonist for the IL‐1 receptor, reversed the effects of IL‐1β, suggesting that the IL‐1β‐dependent up‐regulation of α‐cleavage is mediated by the IL‐1 receptor. IL‐1β also induced this concomitant increase in α‐cleavage and decrease in β‐cleavage in mouse primary cultured neurons. Taken together we conclude that IL‐1β 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.

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+.

Reduced retinal function in amyloid precursor protein-over-expressing transgenic mice via attenuating glutamate-N-methyl-d-aspartate receptor signaling.

Here, we examined whether amyloid‐β (Aβ) protein participates in cell death and retinal function using three types of transgenic (Tg) mice in vivo [human mutant amyloid precursor protein (APP) Tg (Tg 2576) mice, mutant presenilin‐1 (PS‐1) knock‐in mice, and APP/PS‐1 double Tg mice]. ELISA revealed that the insoluble form of Aβ1‐40 was markedly accumulated in the retinas of APP and APP/PS‐1, but not PS‐1 Tg, mice (vs. wild‐type mice). In APP Tg and APP/PS‐1 Tg mice, immunostaining revealed accumulations of intracellular Aβ1–42 in retinal ganglion cells and in the inner and outer nuclear layers. APP Tg and APP/PS‐1 Tg, but not PS‐1 Tg, mice had less NMDA‐induced retinal damage than wild‐type mice, and the reduced damage in APP/PS‐1 Tg mice was diminished by the pre‐treatment of N‐[N‐(3,5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester, a γ‐secretase inhibitor. Furthermore, the number of TUNEL‐positive cells was significantly less in ganglion cell layer of APP/PS‐1 Tg mice than PS‐1 Tg mice 24 h after NMDA injection. The phosphorylated form of calcium/calmodulin‐dependent protein kinase IIα (CaMKIIα), but not total CaMKIIα or total NMDA receptor 1 (NR1) subunit, in total retinal extracts was decreased in non‐treated retinas of APP/PS‐1 Tg mice (vs. wild‐type mice). CaMKIIα and NR2B proteins, but not NR1, in retinal membrane fraction were significantly decreased in APP/PS‐1 Tg mice as compared with wild‐type mice. The NMDA‐induced increase in p‐CaMKIIα in the retina was also lower in APP/PS‐1 Tg mice than in wild‐type mice. In electroretinogram and visual‐evoked potential recordings, the implicit time to each peak from a light stimulus was prolonged in APP/PS‐1 mice versus wild‐type mice. Hence, Aβ may impair retinal function by reducing activation of NMDA‐receptor signaling pathways.

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.

Possible role of scavenger receptor SRCL in the clearance of amyloid‐βin Alzheimer's disease

Accumulation of β‐amyloid protein (Aβ) in the brain is a hallmark of Alzheimers disease (AD), and Aβ‐mediated pathogenesis could result from increased production of Aβ or insufficient Aβ clearance by microglia, astrocytes, or the vascular system. Cell‐surface receptors, such as scavenger receptors, might play a critical role in the binding and clearing of Aβ; however, the responsible receptors have yet to be identified. We show that scavenger receptor with C‐type lectin (SRCL), a member of the scavenger receptor family containing coiled‐coil, collagen‐like, and C‐type lectin/carbohydrate recognition domains, is expressed in cultured astrocytes and microglia. In contrast to the low expression of SRCL in the wild‐type mouse brain, in a double transgenic mouse model of AD (Tg‐APP/PS1), immunohistochemistry showed that SRCL was markedly induced in Aβ‐positive astrocytes and Aβ‐positive vascular/perivascular cells, which are associated closely with cerebral amyloid angiopathy. In patients with AD, the distribution of SRCL was similar to that seen in the Tg‐APP/PS1 temporal cortex. The presence of a large number of SRCL/Aβ double‐positive particles in the intracellular compartments of reactive astrocytes and vascular/perivascular cells in Tg‐APP/PS1 mice and AD patients suggests a role for SRCL in Aβ clearance. Moreover, CHO‐K1 cells transfected with SRCL isoforms were found to bind fibrillar Aβ1–42. These findings suggest that SRCL could be the receptor involved in the binding or clearing of Aβ by glial and vascular/perivascular cells in AD.

Group IB secretory phospholipase A2induces cell death in the cultured cortical neurons: a possible involvement of its binding sites

In primary cultures of rat cortical neurons, group IB secretory phospholipase A(2) (sPLA(2)-IB) induced cell death. In rat cortical membranes, there were high affinity binding sites of [125I]sPLA(2)-IB. The high-affinity binding sites were decreased by sPLA(2)-IB and anti-sPLA(2) receptor immunoglobulin G (anti-sPLA(2)R IgG). Furthermore, anti-sPLA(2)R IgG caused neuronal cell death in a concentration-dependent manner. The present study suggests that sPLA(2)-IB induces neuronal cell death via its high-affinity binding sites.

Deglycosylated anti‐amyloid beta antibodies reduce microglial phagocytosis and cytokine production while retaining the capacity to induce amyloid beta sequestration

Accumulation of amyloid beta (Abeta) is a pathological hallmark of Alzheimers disease, and lowering Abeta is a promising therapeutic approach. Intact anti‐Abeta antibodies reduce brain Abeta through two pathways: enhanced microglial phagocytosis and Abeta transfer from the brain to the periphery (Abeta sequestration). While activation of microglia, which is essential for microglial phagocytosis, is necessarily accompanied by undesired neuroinflammatory events, the capacity for sequestration does not seem to be linked to such effects. We and other groups have found that simple Abeta binding agents are sufficient to reduce brain Abeta through the sequestration pathway. In this study, we aimed to eliminate potentially deleterious immune activation from antibodies without affecting the ability to induce sequestration. The glycan portion of immunoglobulin is critically involved in interactions with immune effectors including the Fc receptor and complement c1q; deglycosylation eliminates these interactions, while antigen (Abeta)‐binding affinity is maintained. In this study, we investigated whether deglycosylated anti‐Abeta antibodies reduce microglial phagocytosis and neuroinflammation without altering the capacity to induce Abeta sequestration. Deglycosylated antibodies maintained Abeta binding affinity. Deglycosylated antibodies did not enhance Abeta phagocytosis or cytokine release in primary cultured microglia, whereas intact antibodies did so significantly. Intravenous injection of deglycosylated antibodies elevated plasma Abeta levels and induced Abeta sequestration to a similar or greater degree compared with intact antibodies in an Alzheimers transgenic mouse model without or with Abeta plaque pathology. We conclude that deglycosylated antibodies effectively induced Abeta sequestration without provoking neuroinflammation; thus, these deglycosylated antibodies may be optimal for sequestration therapy for Alzheimers disease.