Toshiyuki Nakagawa

The Xenopus IP3 receptor: Structure, function, and localization in oocytes and eggs

To study the role of the IP3 receptor (IP3R) upon egg activation, cDNA clones encoding IP3R expressed in the Xenopus oocytes were isolated. By analyses of the primary structure and functional expression of the cDNA, Xenopus IP3R (XIP3R) was shown to have an IP3-binding domain and a putative Ca2+ channel region. Immunocytochemical studies revealed polarized distribution of XIP3R in the cytoplasm of the animal hemisphere in a well-organized endoplasmic reticulum-like structure and intensive localization in the perinuclear region of stage VI immature oocytes. In ovulated unfertilized eggs, XIP3R was densely enriched in the cortical region of both hemispheres in addition to its polarized localization. After fertilization, XIP3R showed a drastic change in its distribution in the cortical region. These results imply the predominant role of the XIP3R in both the formation and propagation of Ca2+ waves at fertilization.

E2-25K/Hip-2 regulates caspase-12 in ER stress–mediated Aβ neurotoxicity

Amyloid-β (Aβ) neurotoxicity is believed to contribute to the pathogenesis of Alzheimers disease (AD). Previously we found that E2-25K/Hip-2, an E2 ubiquitin-conjugating enzyme, mediates Aβ neurotoxicity. Here, we report that E2-25K/Hip-2 modulates caspase-12 activity via the ubiquitin/proteasome system. Levels of endoplasmic reticulum (ER)–resident caspase-12 are strongly up-regulated in the brains of AD model mice, where the enzyme colocalizes with E2-25K/Hip-2. Aβ increases expression of E2-25K/Hip-2, which then stabilizes caspase-12 protein by inhibiting proteasome activity. This increase in E2-25K/Hip-2 also induces proteolytic activation of caspase-12 through its ability to induce calpainlike activity. Knockdown of E2-25K/Hip-2 expression suppresses neuronal cell death triggered by ER stress, and thus caspase-12 is required for the E2-25K/Hip-2–mediated cell death. Finally, we find that E2-25K/Hip-2–deficient cortical neurons are resistant to Aβ toxicity and to the induction of ER stress and caspase-12 expression by Aβ. E2-25K/Hip-2 is thus an essential upstream regulator of the expression and activation of caspase-12 in ER stress–mediated Aβ neurotoxicity.

Autophagy impairment stimulates PS1 expression and gamma-secretase activity.

γ-Secretase plays an important role in the development of Alzheimer disease (AD). γ-Secretase activity is enriched in autophagic vacuoles and it augments amyloid-β (Aβ) synthesis. Autophagy-lysosomal dysfunction has been implicated in AD, but whether γ-secretase activity is affected by autophagy remains unclear. Here we report that γ-secretase activity is enhanced in basal autophagy-disturbed cells through the α subunit of eukaryotic translation initiation factor 2 (eIF2α) kinase, general control nonderepressible 2 (GCN2). Presenilin-1 (PS1) expression was increased even in the presence of nutrients in autophagy-related 5 knockdown (Atg5KD) human embryonic kidney (HEK293) cells expressing a short hairpin RNA as well as in chloroquine-treated HEK293 cells. However, PS1 expression induction was prevented in GCN2KD and ATF4KD cells. Furthermore, Atg5KD cells showed an increase in Aβ production and Notch1 cleavage. These were reduced by an autophagy inducer, resveratrol. Thus, we conclude that the autophagy-lysosomal system regulates γ-secretase activity through GCN2.

Expression and localization of Cayman ataxia-related protein, Caytaxin, is regulated in a developmental- and spatial-dependent manner

Mutation of the gene encoding Caytaxin causes human Cayman ataxia by interfering with normal splicing and, in mutant rodents, by reducing normal transcription, which leads to ataxia, dystonia, and mental retardation: These observations suggest that Caytaxin may be crucial for higher brain functions such as motor learning. We generated antibodies against mouse Caytaxin. Interestingly, we found that the expression of Caytaxin is regulated during brain development while quantitative real time RT-PCR indicated that the mRNA level did not change between postnatal days 7 (P7) and P14 in the cerebellum and hippocampus, implying that the expression of Caytaxin may be controlled by a post-transcriptional mechanism. Immunostaining analyses demonstrated that Caytaxin was localized in many brain areas including the cerebellum and hippocampus. Furthermore, Caytaxin was localized to the presynaptic cytosol by the subcellular fractionation of mouse brain and an observation that was confirmed by co-localization studies with synapsin I and VGLUT1. The above data, disease phenotypes, and mutant animals suggest that Caytaxin may be essential for synaptic function. Thus, identifying the role of Caytaxin in synapse maturation may lead to the development of therapeutic interventions for cerebellar ataxia as well as mental disorders.

Endoplasmic reticulum stress enhances γ-secretase activity.

The endoplasmic reticulum (ER) copes with unfolded proteins in the lumen (ER stress) by activating three distinct intracellular signaling pathways of unfolded protein response (UPR). ER stress contributes to the pathogenesis of obesity and diabetes, which are risk factors for Alzheimers disease (AD) that accelerate the pathogenesis of AD. However, whether ER stress is involved in the development of AD remains unclear. In this study, we demonstrate that ER stress induces presenilin-1 expression through activating transcription factor 4 (ATF4), resulting in increased amyloid-β (Aβ) secretion by γ-secretase activity, which is suppressed by quercetin by modifying UPR signaling. This result suggests that ER stress may be stimulated in obesity and type 2 diabetes, thereby enhancing γ-secretase activity that is the underlying molecular mechanism affecting the pathogenesis of AD.

Epidermal growth factor signaling mediated by grb2 associated binder1 is required for the spatiotemporally regulated proliferation of olig2-expressing progenitors in the embryonic spinal cord.

Gab1 (Grb2 associated binder1) has been identified as an adaptor molecule downstream of many growth factors, including epidermal growth factor (EGF), fibroblast growth factor, and platelet‐derived growth factor, which have been shown to play crucial roles as mitotic signals for a variety of neural progenitor cells, including stem cells, both in vitro and in vivo. Here, we show that Gab1 deficiency results in a reduction in the number of Olig2‐positive (Olig2+) progenitor cells in the developing mouse spinal cord after embryonic day 12.5 (E12.5), when gliogenesis starts in the pMN domain where the EGF receptor (EGFR) is expressed predominantly. Our in vitro analysis further revealed that Gab1 is essential for EGF‐dependent proliferation of Olig2+ progenitor cells derived from the E12.5 ventral and E14.5 dorsal but not ventral spinal cord, whereas Gab1 is always required for the activation of Akt1 but not of ERK1/2. Moreover, we found that the action of the Gab1/Akt pathway is context‐dependent, since constitutively active Akt1 could rescue the proliferation defect only in the E12.5 spinal cord of the Gab1‐deficient mouse in vitro. Finally, we demonstrated that EGFR‐deficient mice and Gab1‐deficient mice showed a similar reduction in the number of Olig2+ progenitor cells in the developing spinal cord. These findings indicate that EGFR‐mediated signaling through Gab1/Akt contributes to the sufficient expansion of Olig2+ progenitor cells in a spatiotemporally regulated manner, which represents the origin of glial cells in the developing spinal cord.

Cayman Ataxia-Related Protein is a Presynapse-Specific Caspase-3 Substrate

Caspase plays an important role in apoptosis and physiological processes such as synaptic plasticity. However, the caspase substrate at the synapse is still unknown. Here we used an in vitro cleavage assay with a small-pool human brain cDNA library. We identified the presynaptic protein Caytaxin as a substrate of caspase-3 and caspase-7. Deficiency in Caytaxin causes Cayman ataxia, a disorder characterized by cerebellar dysfunction and mental retardation. Caytaxin cleavage in cerebellar granule neurons is dependent on caspase-3 activation. The cleavage site is upstream of the cellular retinal and the TRIO guanine exchange factor domain, producing a C-terminal fragment that may play an alternative role in inhibiting MEK2 signaling. Thus, we concluded that Caytaxin is a novel substrate of caspase-3 at the presynapse.

Polyglutamine expansion disturbs the endoplasmic reticulum formation, leading to caspase-7 activation through Bax

The endoplasmic reticulum (ER) plays a pivotal role in cellular functions such as the ER stress response. However, the effect of the ER membrane on caspase activation remains unclear. This study reveals that polyglutamine oligomers augmented at ER induce insertion of Bax into the ER membrane, thereby activating caspase-7. In line with the role of ER in cell death induced by polyglutamine expansion, the ER membrane was found to be disrupted and dilated in the brain of a murine model of Huntingtons disease. We can conclude that polyglutamine expansion may drive caspase-7 activation by disrupting the ER membrane.

OCIAD2 activates γ-secretase to enhance amyloid β production by interacting with nicastrin

The gamma (γ)-secretase holoenzyme is composed of four core proteins and cleaves APP to generate amyloid beta (Aβ), a key molecule that causes major neurotoxicity during the early stage of Alzheimer’s disease (AD). However, despite its important role in Aβ production, little is known about the regulation of γ-secretase. OCIAD2, a novel modulator of γ-secretase that stimulates Aβ production, and which was isolated from a genome-wide functional screen using cell-based assays and a cDNA library comprising 6,178 genes. Ectopic expression of OCIAD2 enhanced Aβ production, while reduction of OCIAD2 expression suppressed it. OCIAD2 expression facilitated the formation of an active γ-secretase complex and enhanced subcellular localization of the enzyme components to lipid rafts. OCIAD2 interacted with nicastrin to stimulate γ-secretase activity. OCIAD2 also increased the interaction of nicastrin with C99 and stimulated APP processing via γ-secretase activation, but did not affect Notch processing. In addition, a cell-permeable Tat-OCIAD2 peptide that interfered with the interaction of OCIAD2 with nicastrin interrupted the γ-secretase-mediated AICD production. Finally, OCIAD2 expression was significantly elevated in the brain of AD patients and PDAPP mice. This study identifies OCIAD2 as a selective activator of γ-secretase to increase Aβ generation.

The expression and localization of Prune2 mRNA in the central nervous system

A family of Bcl-2/adenovirus E1B 19kDa-interacting proteins (BNIPs) plays critical roles in several cellular processes such as cellular transformation, apoptosis, neuronal differentiation, and synaptic function, which are mediated by the BNIP2 and Cdc42GAP homology (BCH) domain. Prune homolog 2 (Drosophila) (PRUNE2) and its isoforms -C9orf65, BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1), and BNIP2 Extra Long (BNIPXL) - have been shown to be a susceptibility gene for Alzheimers disease, a biomarker for leiomyosarcomas, a proapoptotic protein in neuronal cells, and an antagonist of cellular transformation, respectively. However, precise localization of PRUNE2 in the brain remains unclear. Here, we identified the distribution of Prune2 mRNA in the adult mouse brain. Prune2 mRNA is predominantly expressed in the neurons of the cranial nerve motor nuclei and the motor neurons of the spinal cord. The expression in the dorsal root ganglia (DRG) is consistent with the previously described reports. In addition, we observed the expression in another sensory neuron in the mesencephalic trigeminal nucleus. These results suggest that Prune2 may be functional in these restricted brain regions.