Naohiro Itoh

Cdc42 and Rac1 Regulate the Interaction of IQGAP1 with β-Catenin

IQGAP1, a target of Cdc42 and Rac1 small GTPases, directly interacts with β-catenin and negatively regulates E-cadherin-mediated cell-cell adhesion by dissociating α-catenin from the cadherin-catenin complex in vivo (Kuroda, S., Fukata, M., Nakagawa, M., Fujii, K., Nakamura, T., Ookubo, T., Izawa, I., Nagase, T., Nomura, N., Tani, H., Shoji, I., Matsuura, Y., Yonehara, S., and Kaibuchi, K. (1998) Science 281, 832–835). Here we investigated how Cdc42 and Rac1 regulate the IQGAP1 function. IQGAP1 interacted with the amino-terminal region (amino acids 1–183) of β-catenin, which contains the α-catenin-binding domain. IQGAP1 dissociated α-catenin from the β-catenin-α-catenin complex in a dose-dependent manner in vitro. Guanosine 5′-(3-O-thio)triphosphate (GTPγS)·glutathioneS-transferase (GST)-Cdc42 and GTPγS·GST-Rac1 inhibited the binding of IQGAP1 to β-catenin in a dose-dependent manner in vitro, whereas neither GDP·GST-Cdc42, GDP·GST-Rac1, nor GTPγS·GST-RhoA did. The coexpression of dominant active Cdc42 with IQGAP1 suppressed the dissociation of α-catenin from the cadherin-catenin complex induced by the overexpression of IQGAP1 in L cells expressing E-cadherin (EL cells). Consistent with this, the overexpression of either dominant negative Cdc42 or Rac1 resulted in the reduction of E-cadherin-mediated cell adhesive activity in EL cells. These results indicate that Cdc42 and Rac1 negatively regulate the IQGAP1 function by inhibiting the interaction of IQGAP1 with β-catenin, leading to stabilization of the cadherin-catenin complex.

Regulation of Notch Signaling by Dynamic Changes in the Precision of S3 Cleavage of Notch-1†

ABSTRACT Intramembrane proteolysis by presenilin-dependent γ-secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-β. Here, we show that upon Notch signaling the intracellular domain of Notch-1 is cleaved into two distinct types of NICD species due to diversity in the site of S3 cleavage. Consistent with the N-end rule, the S3-V cleavage produces stable NICD with Val at the N terminus, whereas the S3-S/S3-L cleavage generates unstable NICD with Ser/Leu at the N terminus. Moreover, intracellular Notch signal transmission with unstable NICDs is much weaker than that with stable NICD. Importantly, the extent of endocytosis in target cells affects the relative production ratio of the two types of NICD, which changes in parallel with Notch signaling. Surprisingly, substantial amounts of unstable NICD species are generated from the Val→Gly and the Lys→Arg mutants, which have been reported to decrease S3 cleavage efficiency in cultured cells. Thus, we suggest that the existence of two distinct types of NICD points to a novel aspect of the intracellular signaling and that changes in the precision of S3 cleavage play an important role in the process of conversion from extracellular to intracellular Notch signaling.

Secretion of the Notch-1 Aβ-like peptide during Notch signaling

The canonical pathway of Notch signaling is mediated by regulated intramembrane proteolysis (RIP). In the pathway, ligand binding results in sequential proteolysis of the Notch receptor, and presenilin (PS)-dependent intramembrane proteolysis at the interface between the membrane and cytosol liberates the Notch-1 intracellular domain (NICD), a transcription modifier. Because the degradation of the Notch-1 transmembrane domain is thought to require an additional cleavage near the middle of the transmembrane domain, extracellular small peptides (Notch-1 Aβ-like peptide (Nβ)) should be produced. Here we showed that Nβ species are indeed secreted during the process of Notch signaling. We identified mainly two distinct molecular species of novel Nβ, Nβ21 and C-terminally elongated Nβ25, which were produced in an ∼5:1 ratio. This process is reminiscent of the production of Alzheimer disease-associated Aβ. PS pathogenic mutants increased the production of the longer species of Aβ (Aβ42) from β-amyloid protein precursor. We revealed that several Alzheimer disease mutants also cause a parallel increase in the secretion of the longer form of Nβ. Strikingly, chemicals that modify the Aβ42 level caused parallel changes in the Nβ25 level. These results demonstrated that the characteristics of C-terminal elongation of Nβ and Aβ are almost identical. In addition, because many other type 1 membrane-bound receptors release intracellular domains by PS-dependent intramembrane proteolysis, we suspect that the release of Aβ- or Nβ-like peptides is a common feature of the proteolysis during RIP signaling. We anticipate that this study will open the door to searches for markers of RIP signaling and surrogate markers for Aβ42 production.

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+.

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.

Novel binding sites of 15-deoxy-Δ12,14-prostaglandin J2 in plasma membranes from primary rat cortical neurons

15-Deoxy-Delta12,14-prostaglandin J2 (15d-Delta12,14-PGJ2) is an endogenous ligand for a nuclear peroxysome proliferator activated receptor-gamma (PPAR). We found novel binding sites of 15d-Delta12,14-PGJ2 in the neuronal plasma membranes of the cerebral cortex. The binding sites of [3H]15d-Delta12,14-PGJ2 were displaced by 15d-Delta12,14-PGJ2 with a half-maximal concentration of 1.6 microM. PGD2 and its metabolites also inhibited the binding of [3H]15d-Delta12,14-PGJ2. Affinities for the novel binding sites were 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. Other eicosanoids and PPAR agonists did not alter the binding of [3H]15d-Delta12,14-PGJ2. In primary cultures of rat cortical neurons, we examined the pathophysiologic roles of the novel binding sites. 15d-Delta12,14-PGJ2 triggered neuronal cell death in a concentration-dependent manner, with a half-maximal concentration of 1.1 microM. The neurotoxic potency of PGD2 and its metabolites was also 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. The morphologic and ultrastructural characteristics of 15d-Delta12,14-PGJ2-induced neuronal cell death were apoptotic, as evidenced by condensed chromatin and fragmented DNA. On the other hand, we detected little neurotoxicity of other eicosanoids and PPAR agonists. In conclusion, we demonstrated that novel binding sites of 15d-Delta12,14-PGJ2 exist in the plasma membrane. The present study suggests that the novel binding sites might be involved in 15d-Delta12,14-PGJ2-induced neuronal apoptosis.

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.

Transcriptome analysis of distinct mouse strains reveals kinesin light chain-1 splicing as an amyloid-β accumulation modifier

Significance Genetic studies of common complex human diseases, including Alzheimers disease (AD), are extremely resource-intensive and have struggled to identify genes that are causal in disease. Combined with the costs of studies and the inability to identify the missing heritability, particularly in AD, alternate strategies warrant consideration. We devised a unique strategy that combines distinct mouse strains that vary naturally in amyloid-β production with transcriptomics to identify kinesin light chain-1 (Klc1) splice variant E as a modifier of amyloid-β accumulation, a causative factor of AD. In AD patients, the expression levels of KLC1 variant E in brain were significantly higher compared with levels in unaffected individuals. The identification of KLC1 variant E suggests that dysfunction of intracellular trafficking is causative in AD. Alzheimer’s disease (AD) is characterized by the accumulation of amyloid-β (Aβ). The genes that govern this process, however, have remained elusive. To this end, we combined distinct mouse strains with transcriptomics to directly identify disease-relevant genes. We show that AD model mice (APP-Tg) with DBA/2 genetic backgrounds have significantly lower levels of Aβ accumulation compared with SJL and C57BL/6 mice. We then applied brain transcriptomics to reveal the genes in DBA/2 that suppress Aβ accumulation. To avoid detecting secondarily affected genes by Aβ, we used non-Tg mice in the absence of Aβ pathology and selected candidate genes differently expressed in DBA/2 mice. Additional transcriptome analysis of APP-Tg mice with mixed genetic backgrounds revealed kinesin light chain-1 (Klc1) as an Aβ modifier, indicating a role for intracellular trafficking in Aβ accumulation. Aβ levels correlated with the expression levels of Klc1 splice variant E and the genotype of Klc1 in these APP-Tg mice. In humans, the expression levels of KLC1 variant E in brain and lymphocyte were significantly higher in AD patients compared with unaffected individuals. Finally, functional analysis using neuroblastoma cells showed that overexpression or knockdown of KLC1 variant E increases or decreases the production of Aβ, respectively. The identification of KLC1 variant E suggests that the dysfunction of intracellular trafficking is a causative factor of Aβ pathology. This unique combination of distinct mouse strains and model mice with transcriptomics is expected to be useful for the study of genetic mechanisms of other complex diseases.