© 2019 The Author(s) Cereblon (CRBN) is a substrate recognition subunit of the CRL4 E3 ubiquitin ligase complex, directly binding to specific substrates for poly-ubiquitination followed by proteasome-dependent degradation of proteins. Cellular CRBN is responsible for energy metabolism, ion-channel activation, and cellular stress response through binding to proteins related to the respective pathways. As CRBN binds to various proteins, the selective pressure at the interacting surface is expected to result in functional divergence. Here, we present two mammalian CRBN datasets of molecular evolutionary analyses. (1) The multiple sequence alignment data shows that positive selection occurred, determined with a dN/dS calculation. (2) Data on co-evolutionary analysis between vertebrate CRBN and related proteins are represented by calculating the correlation coefficient based on the comparison of phylogenetic trees. Co-evolutionary analysis shows the similarity of evolutionary traits of two proteins. Further molecular, functional interpretation of these analyses is explained in ‘Positive selection of Cereblon modified function including its E3 Ubiquitin Ligase activity and binding efficiency with AMPK’ (W. Onodera, T. Asahi, N. Sawamura, Positive selection of cereblon modified function including its E3 ubiquitin ligase activity and binding efficiency with AMPK. Mol Phylogenet Evol. (2019) 135:78-85. [1]).

Thalidomide was originally used as a sedative and found to be a teratogen, but now thalidomide and its derivatives are widely used to treat haematologic malignancies. Accumulated evidence suggests that thalidomide suppresses nerve cell death in neurologic model mice. However, detailed molecular mechanisms are unknown. Here we examined the molecular mechanism of thalidomide's neuroprotective effects, focusing on its target protein, cereblon (CRBN), and its binding protein, AMP-activated protein kinase (AMPK), which plays an important role in maintaining intracellular energy homeostasis in the brain. We used a cerebral ischemia rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). Thalidomide treatment significantly decreased the infarct volume and neurological deficits of MCAO/R rats. AMPK was the key signalling protein in this mechanism. Furthermore, we considered that the AMPK-CRBN interaction was altered when neuroprotective action by thalidomide occurred in cells under ischemic conditions. Binding was strong between AMPK and CRBN in normal SH-SY5Y cells, but was weakened by the addition of H2O2. However, when thalidomide was administered at the same time as H2O2, the binding of AMPK and CRBN was partly restored. These results suggest that thalidomide inhibits the activity of AMPK via CRBN under oxidative stress and suppresses nerve cell death.

Ge-132 is a synthetic organic germanium that is used as a dietary supplement. The antioxidant activity of Ge-132 on cultured mammalian cells was investigated in this study. First, Ge-132 cytotoxicity on mammalian cultured cells was determined by measuring lactate dehydrogenase (LDH) levels. Ge-132 had no cytotoxic effect on three different cell lines. Second, the cell proliferative effect of Ge-132 was determined by measuring ATP content of whole cells and counting them. Ge-132 treatment of Chinese hamster ovary (CHO-K1) and SH-SY5Y cells promoted cell proliferation in a dose-dependent manner. Finally, antioxidant activity of Ge-132 against hydrogen peroxide-induced oxidative stress was determined by measuring the levels of intracellular reactive oxygen species (ROS) and carbonylated proteins. Pre-incubation of CHO-K1 and SH-SY5Y cells with Ge-132 suppressed intracellular ROS production and carbonylated protein levels induced by hydrogen peroxide. Our results suggest that Ge-132 has antioxidant activity against hydrogen peroxide-induced oxidative stress.

Amyloid 13 protein (A beta) plays a central role in Alzheimer's disease (AD) pathogenesis. Point mutations in the A beta sequence, which cluster around the central hydrophobic core of the peptide, are associated with familial AD (FAD). Several mutations have been identified, with the Arctic mutation exhibiting a purely cognitive phenotype that is typical of AD. Our previous findings suggest that Arctic A beta 40 binds to and aggregates with CHRNA7, thereby inhibiting the calcium response and signaling pathways downstream of the receptor. Activation of CHRNA7 is neuroprotective both in vitro and in vivo. Therefore, in the present study, we investigated whether Arctic A beta 40 affects neuronal survival and/or death via CHRNA7. Using human neuroblastoma SH-SY5Y cells, we found that the neuroprotective function of CHRNA7 is blocked by CHRNA7 knockdown using RNA interference. Furthermore, Arctic A beta 40 blocked the neuroprotective effect of nicotine by inhibiting the ERK1/2 pathway downstream of CHRNA7. Moreover, we show that ERK1/2 activation mediates the neuroprotective effect of nicotine against oxidative stress. Collectively, our findings further our understanding of the molecular pathogenesis of Arctic FAD. (C) 2017 Elsevier Ltd. All rights reserved.

Amyloid β protein (Aβ) plays a central role in Alzheimer's disease (AD) pathogenesis. Point mutations in the Aβ sequence, which cluster around the central hydrophobic core of the peptide, are associated with familial AD (FAD). Several mutations have been identified, with the Arctic mutation exhibiting a purely cognitive phenotype that is typical of AD. Our previous findings suggest that Arctic Aβ40 binds to and aggregates with CHRNA7, thereby inhibiting the calcium response and signaling pathways downstream of the receptor. Activation of CHRNA7 is neuroprotective both in vitro and in vivo. Therefore, in the present study, we investigated whether Arctic Aβ40 affects neuronal survival and/or death via CHRNA7. Using human neuroblastoma SH-SY5Y cells, we found that the neuroprotective function of CHRNA7 is blocked by CHRNA7 knockdown using RNA interference. Furthermore, Arctic Aβ40 blocked the neuroprotective effect of nicotine by inhibiting the ERK1/2 pathway downstream of CHRNA7. Moreover, we show that ERK1/2 activation mediates the neuroprotective effect of nicotine against oxidative stress. Collectively, our findings further our understanding of the molecular pathogenesis of Arctic FAD.

Cereblon (CRBN) is a substrate receptor of the E3 ubiquitin ligase complex that is highly conserved in animals and plants. CRBN proteins have been implicated in various biological processes such as development, metabolism, learning, and memory formation, and their impairment has been linked to autosomal recessive non-syndromic intellectual disability and cancer. Furthermore, human CRBN was identified as the primary target of thalidomide teratogenicity. Data on functional analysis of CRBN family members in vivo, however, are still scarce. Here we identify Ohgata (OHGT), the Drosophila ortholog of CRBN, as a regulator of insulin signaling-mediated growth. Using ohgt mutants that we generated by targeted mutagenesis, we show that its loss results in increased body weight and organ size without changes of the body proportions. We demonstrate that ohgt knockdown in the fat body, an organ analogous to mammalian liver and adipose tissue, phenocopies the growth phenotypes. We further show that overgrowth is due to an elevation of insulin signaling in ohgt mutants and to the down-regulation of inhibitory cofactors of circulating Drosophila insulin-like peptides (DILPs), named acid-labile subunit and imaginal morphogenesis protein-late 2. The two inhibitory proteins were previously shown to be components of a heterotrimeric complex with growth-promoting DILP2 and DILP5. Our study reveals OHGT as a novel regulator of insulin-dependent organismic growth in Drosophila.

The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. (C) 2016 Elsevier Inc. All rights reserved.

Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we determined if CRBN has a protective function against oxidative stress, similar to Lon protease. We report that CRBN partially distributes in mitochondria, suggesting it has a mitochondrial function. To specify the mitochondrial role of CRBN, we mitochondrially expressed CRBN in human neuroblastoma SH-SY5Y cells. The resulting stable SH-SY5Y cell line showed no apparent effect on the mitochondrial functions of fusion, fission, and membrane potential. However, mitochondrially expressed CRBN exhibited protease activity, and was induced by oxidative stress. In addition, stably expressed cells exhibited suppressed neuronal cell death induced by hydrogen peroxide. These results suggest that CRBN functions specifically as a Lon-type protease in mitochondria.

Cereblon (CRBN) is encoded by a candidate gene for autosomal recessive nonsyndromic intellectual disability (ID). The nonsense mutation, R419X, causes deletion of 24 amino acids at the C-terminus of CRBN, leading to mild ID. Although abnormal CRBN function may be associated with ID disease onset, its cellular mechanism is still unclear. Here, we examine the role of CRBN in aggresome formation and cytoprotection. In the presence of a proteasome inhibitor, exogenous CRBN formed perinuclear inclusions and co-localized with aggresome markers. Endogenous CRBN also formed perinuclear inclusions under the same condition. Treatment with a microtubule destabilizer or an inhibitor of the E3 ubiquitin ligase activity of CRBN blocked formation of CRBN inclusions. Biochemical analysis showed CRBN containing inclusions were high-molecular weight, ubiquitin-positive. CRBN overexpression in cultured cells suppressed cell death induced by proteasome inhibitor. Furthermore, knockdown of endogenous CRBN in cultured cells increased cell death induced by proteasome inhibitor, compared with control cells. Our results show CRBN is recruited to aggresome and has functional roles in cytoprotection against ubiquitin-proteasome system impaired condition.

Cereblon (CRBN) is encoded by a candidate gene for autosomal recessive nonsyndromic intellectual disability (ID). The nonsense mutation, R419X, causes deletion of 24 amino acids at the C-terminus of CRBN, leading to mild ID. Although abnormal CRBN function may be associated with ID disease onset, its cellular mechanism is still unclear. Here, we examine the role of CRBN in aggresome formation and cytoprotection. In the presence of a proteasome inhibitor, exogenous CRBN formed perinuclear inclusions and co-localized with aggresome markers. Endogenous CRBN also formed perinuclear inclusions under the same condition. Treatment with a microtubule destabilizer or an inhibitor of the E3 ubiquitin ligase activity of CRBN blocked formation of CRBN inclusions. Biochemical analysis showed CRBN containing inclusions were high-molecular weight, ubiquitin-positive. CRBN overexpression in cultured cells suppressed cell death induced by proteasome inhibitor. Furthermore, knockdown of endogenous CRBN in cultured cells increased cell death induced by proteasome inhibitor, compared with control cells. Our results show CRBN is recruited to aggresome and has functional roles in cytoprotection against ubiquitin-proteasome system impaired condition. (C) 2015 Elsevier Inc. All rights reserved.

Amyloid protein (A beta) plays a central role in the pathogenesis of Alzheimer's disease (AD). Point mutations within the A sequence associated with familial AD (FAD) are clustered around the central hydrophobic core of A. Several types of mutations within the A sequence have been identified, and the Arctic' mutation (E22G) has a purely cognitive phenotype typical of AD. Previous studies have shown that the primary result of the Arctic' mutation is increased formation of A protofibrils. However, the molecular mechanism underlying this effect remains unknown. A42 binds to a neuronal nicotinic acetylcholine receptor subunit, neuronal acetylcholine receptor subunit alpha-7 (CHRNA7), with high affinity and, thus, may be involved in the pathogenesis of AD. Therefore, to clarify the molecular mechanism of Arctic mutation-mediated FAD, we focused on CHRNA7 as a target molecule of Arctic A. We performed an in vitro binding assay using purified CHRNA7 and synthetic Arctic A40, and demonstrated that Arctic A40 specifically bound to CHRNA7. The aggregation of Arctic A40 was enhanced with the addition of CHRNA7. Furthermore, the function of CHRNA7 was detected by measuring Ca2+ flux and phospho-p44/42 MAPK (ERK1/2) activation. Our results indicated that Arctic A40 aggregation was enhanced by the addition of CHRNA7, which destabilized the function of CHRNA7 via inhibition of Ca2+ responses and activation of ERK1/2. These findings indicate that Arctic A mutation may be involved in the mechanism underlying FAD. This mechanism may involve binding and aggregation, leading to the inhibition of CHRNA7 functions.

We propose, as an alternative to conventional spectroscopic assays, a simple method for discriminating fibrous amyloid proteins by using a label-free semiconductor-based biosensor. The highly sensitive assay is expected to be useful for accelerating amyloid related research.

The proton motive force (PMF) is bio-energetically important for various cellular reactions to occur. We developed PMF-photogenerating mitochondria in cultured mammalian cells. An archaebacterial rhodopsin, delta-rhodopsin, which is a light-driven proton pump derived from Haloterrigena turkmenica, was expressed in the mitochondria of CHO-K1 cells. The constructed stable CHO-K1 cell lines showed suppression of cell death induced by rotenone, a pesticide that inhibits mitochondrial complex I activity involved in PMF generation through the electron transport chain. Delta-rhodopsin was also introduced into the mitochondria of human neuroblastoma SH-SY5Y cells. The constructed stable SH-SY5Y cell lines showed suppression of dopaminergic neuronal cell death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an inducer of Parkinson's disease models, which acts through inhibition of complex I activity. These results suggest that the light-activated proton pump functioned as a PMF generator in the mitochondria of mammalian cells, and suppressed cell death induced by inhibition of respiratory PMF generation.

Disrupted-in-schizophrenia-1 (DISC1) is one of major susceptibility factors for a wide range of mental illnesses, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions. DISC1 is located in several subcellular domains, such as the centrosome and the nucleus, and interacts with various proteins, including NudE-like (NUDEL/NDEL1) and activating transcription factor 4 (ATF4)/CREB2. Nevertheless, a role for DISC1 in vivo remains to be elucidated. Therefore, we have generated a Drosophila model for examining normal functions of DISC1 in living organisms. DISC1 transgenic flies with preferential accumulation of exogenous human DISC1 in the nucleus display disturbance in sleep homeostasis, which has been reportedly associated with CREB signaling/CRE-mediated gene transcription. Thus, in mammalian cells, we characterized nuclear DISC1, and identified a subset of nuclear DISC1 that colocalizes with the promyelocytic leukemia (PML) bodies, a nuclear compartment for gene transcription. Furthermore, we identified three functional cis-elements that regulate the nuclear localization of DISC1. We also report that DISC1 interacts with ATF4/CREB2 and a corepressor N-CoR, modulating CRE-mediated gene transcription.

Here we analyze the species conservation of disrupted-in-schizophrenia-1 (DISC1) gene, a susceptibility gene for schizophrenia. We cloned cDNA of DISCI and characterized DISCI protein in monkey brains and compared their features with those in a variety of species, including humans, rodents and lower vertebrates. Sequences of human and monkey DISCI are very similar for both nucleotides and amino acids, in sharp contrast to those of rodents; this is reminiscent of G72, another gene involved in major mental illnesses. Bioinformatic cross-species comparisons identified a portion of DISC] sequences in chicken and Caenorhabditis elegans, but failed to find DISC1 in Drosophila. In contrast to sequence differences, the regional expression profile of DISCI is well conserved between rodents and primates in that levels of DISC I mRNA and protein are higher in the hippocampus and the cerebral cortex, and much lower in cerebellum in adult brains. The findings of this study may suggest overall patterns of evolution of genes for psychiatric disorders, and thus assist in production of genetically-engineered mice, and the interpretation of the underlying mechanisms of psychiatric conditions. Published by Elsevier Ireland Ltd on behalf of Japan Neuroscience Society.

Here we overview Disrupted-in-Schizophrenia-1 (DISC1), a promising lead in studying the pathophysiology of major mental conditions. Genetic association studies reproducibly suggest involvement of DISC1 in both schizophrenia and bipolar disorder in several ethnic groups. Different from several other susceptibility genes for schizophrenia, such as neuregulin-1 and dysbindin, there are two independent pedigrees in which genetic variations of DISC1 directly segregate with major mental conditions. This uniqueness has facilitated neurobiology of DISC1, which may hopefully lead to an important breakthrough in understanding of pathophysiology of major mental conditions. DISC1 is a multifunctional protein that plays a role in neurodevelopment and cell signaling. In autopsied brains from patients with psychosis and substance abuse, change in subcellular distribution of DISC1 is observed. DISC1 interacts with phosphodiesterase (PDE) 4B that degrades cyclic AMP (cAMP), which may be a regulatory molecule for working memory in the prefrontal cortex. Knockdown expression of DISC1 in developing cerebral cortex in mouse brains leads to changes that resemble, at least in part, the pathology found in patients with schizophrenia. These results support involvement of DISC1 in the pathophysiology of major mental conditions, including schizophrenia, in several mechanisms.

統合失調症の病態に複数の遺伝子と環境要因の関与が指摘されている.最近連鎖研究・関連研究からいくつかの統合失調症の候補遺伝子が明らかになってきた.本稿では,そのなかからDISC1(Disrupted-In-Schizophrenia 1)からみた統合失調症の病態研究を展望する.DISC1は精神疾患多発単一家系から発見されたが,遺伝学的研究と統合失調症患者剖検脳を用いた生化学的研究から統合失調症一般例においてもDISC1の統合失調症への関与が示唆されている.DISC1は多くの細胞内部位で働く多機能タンパクであり,特に細胞骨格に関与して神経発達に重要な役割を果たしている.DISC1のさらなる解析,特にDISC1のトランスジェニックマウスによる統合失調症の動物モデルの確立が統合失調症の病態理解や治療法の開発につながることが期待される(著者抄録)

Disrupted-In-Schizophrenia-1 (DISC1), originally identified at the breakpoint of a chromosomal translocation that is linked to a rare familial schizophrenia, has been genetically implicated in schizophrenia in other populations. Schizophrenia involves subtle cytoarchitectural abnormalities that arise during neurodevelopment, but the underlying molecular mechanisms are unclear. Here, we demonstrate that DISC1 is a component of the microtubule-associated dynein motor complex and is essential for maintaining the complex at the centrosome, hence contributing to normal microtubular dynamics. Carboxy-terminal-truncated mutant DISC1 (mutDISC1), which results from a chromosomal translocation, functions in a dominant-negative manner by redistributing wildtype DISC1 through self-association and by dissociating the DISC1-dynein complex from the centrosome. Consequently, either depletion of endogenous DISC1 or expression of mutDISC1 impairs neurite outgrowth in vitro and proper development of the cerebral cortex in vivo. These results indicate that DISC1 is involved in cerebral cortex development, and suggest that loss of DISCI function may underlie neurodevelopmental dysfunction in schizophrenia.

Although the genetics of schizophrenia (SZ) remains unresolved, recently both linkage/association studies and cytogenetic approaches have clarified several possible genes for SZ. In the neuropathology of SZ, many now agree that there are at least cytoarchitectural abnormalities such as alterations in synaptic, dendritic, and axonal organization, but clear molecular markers unique to SZ are still missing.
In this review, we propose an approach relying on Disrupted-In-Schizophrenia-1 (DISC1), a candidate gene for SZ that may shed light on the molecular neuropathology of SZ. DISC1 was originally identified as the sole disrupted gene with an open reading frame, from a 'unique' Scottish family in which familial SZ and other major mental illnesses occur in tight association with a hereditary chromosomal abnormality. Additional genetic and biochemical approaches using autopsied brains from patients with SZ from other populations than the Scottish family suggest that DISC1 may be implicated not only in the Scottish family but also in general' cases of SZ. Furthermore. functional dissection of DISC1 protein indicates that DISC1 is a cytoskeletal protein that plays a key role in neurodevelopment, which may be relevant to the pathogenesis of SZ.
Here, we provide a summary of the current updates of studies of DISC1 and their future perspective, especially its possible role in the pathophysiology of SZ, in association with other genetic and environmental factors. (c) 2005 Association for Research in Nervous and Mental Disease. Published by Elsevier B.V. All rights reserved.

Disrupted-In-Schizophrenia 1 (DISC1) was identified as the sole gene whose ORF is truncated and cosegregates with major mental illnesses in a Scottish family. DISC1 has also been suggested, by association and linkage studies, to be a susceptibility gene for schizophrenia (SZ) in independent populations. However, no analysis of DISC1 protein in human brains, especially those of patients with SZ, has yet been conducted. Here we performed a biochemical analysis of DISC1 protein in a well characterized set of autopsied brains, including brains of patients with SZ, bipolar disorder, and major depression (MD), as well as normal control brains. We identified an isoform of DISC1 by using MS and demonstrated that it is enriched in the nucleus of HeLa cells. In the orbitofrontal cortex, the subcellular distribution of this DISC1 isoform, assessed by the nuclear to cytoplasmic ratio in the immunoreactivity of the isoform, is significantly changed in brains from patients with SZ and MD. This altered distribution is also observed in those subjects with substance and alcohol abuse. The changes in MD brains are significantly influenced by substance/alcohol abuse as well as postmortem interval; however, the alteration in SZ brains is free from brain-associated confounding factors, although an interaction with substance/alcohol abuse cannot be completely ruled out. These results suggest that DISC1 may be implicated in psychiatric conditions in other populations than the unique Scottish family.

Lipid rafts and their component, cholesterol, modulate the processing of beta-amyloid precursor protein (APP). However, the role of sphingolipids, another major component of lipid rafts, in APP processing remains undetermined. Here we report the effect of sphingolipid deficiency on APP processing in Chinese hamster ovary cells treated with a specific inhibitor of serine palmitoyltransferase, which catalyzes the first step of sphingolipid biosynthesis, and in a mutant LY-B strain defective in the LCB1 subunit of serine palmitoyltransferase. We found that in sphingolipid-deficient cells, the secretion of soluble APPalpha (sAPPalpha) and the generation of C-terminal fragment cleaved at alpha-site dramatically increased, whereas epsilon-cleavage activity remained unchanged, and the epsilon-cleavage activity decreased without alteration of the total APP level. The secretion of amyloid beta-protein 42 increased in sphingolipid-deficient cells, whereas that of amyloid beta-protein 40 did not. All of these alterations were restored in sphingolipid-deficient cells by adding exogenous sphingosine and in LY-B cells by transfection with cLCB1. Sphingolipid deficiency increased MAPK/ERK activity and a specific inhibitor of MAPK kinase, PD98059, restored sAPPalpha level, indicating that sphingolipid deficiency enhances sAPPalpha secretion via activation of MAPK/ERK pathway. These results suggest that not only the cellular level of cholesterol but also that of sphingolipids may be involved in the pathological process of Alzheimer's disease by modulating APP cleavage.

統合失調症は思春期に好発する難治性の精神疾患である.その有病率は人口の1%に及び,継続的な医療が必要にも拘わらず,社会復帰や根治が困難であることから,その病態の把握は急務となっている.こうした中,遺伝学的検索から発見されたDisrupted-In-Schizophrenia 1(DISC1)は,精神疾患研究でおそらく最初にみとめられた候補遺伝子であり,このDISC1及びその遺伝子産物の解析を進めることにより,その病態に迫ることができるのではないかと現在期待されている

Previously, we found that amyloid beta-protein (Abeta)1-42 exhibits neurotoxicity, while Abeta1-40 serves as an antioxidant molecule by quenching metal ions and inhibiting metal-mediated oxygen radical generation. Here, we show another neuroprotective action of nonamyloidogenic Abeta1-40 against Abeta1-42-induced neurotoxicity in culture and in vivo. Neuronal death was induced by Abeta1-42 at concentrations higher than 2 mum, which was prevented by concurrent treatment with Abeta1-40 in a dose-dependent manner. However, metal chelators did not prevent Abeta1-42-induced neuronal death. Circular dichroism spectroscopy showed that Abeta1-40 inhibited the beta-sheet transformation of Abeta1-42. Thioflavin-T assay and electron microscopy analysis revealed that Abeta1-40 inhibited the fibril formation of Abeta1-42. In contrast, Abeta1-16, Abeta25-35, and Abeta40-1 did not inhibit the fibril formation of Abeta1-42 nor prevent Abeta1-42-induced neuronal death. Abeta1-42 injection into the rat entorhinal cortex (EC) caused the hyperphosphorylation of tau on both sides of EC and hippocampus and increased the number of glial fibrillary acidic protein (GFAP)-positive astrocytes in the ipsilateral EC, which were prevented by the concurrent injection of Abeta1-40. These results indicate that Abeta1-40 protects neurons from Abeta1-42-induced neuronal damage in vitro and in vivo, not by sequestrating metals, but by inhibiting the beta-sheet transformation and fibril formation of Abeta1-42. Our data suggest a mechanism by which elevated Abeta1-42/Abeta1-40 ratio accelerates the development of Alzheimer's disease (AD) in familial AD.

Niemann-Pick type C (NPC) disease is a cholesterol-storage disease accompanied by neurodegeneration with the formation of neurofibrillary tangles, the major component of which is the hyperphosphorylated tau. Here, we examined the mechanism underlying hyperphosphorylation of tau using mutant Chinese hamster ovary (CHO) cell line defective in NPC1 (CT43) as a tool. Immunoblot analysis revealed that tau was hyperphosphorylated at multiple sites in CT43 cells, but not in their parental cells (25RA) or the wild-type CHO cells. In CT43 cells, mitogen-activated protein (MAP) kinase Erk1/2 was activated and the specific MAPK inhibitor, PD98059, attenuated the hyperphosphorylation of tau. The amount of protein phosphatase 2A not bound to microtubules was decreased in CT43 cells. CT43 cells but not 25RA cells were amphotericin B-resistant, indicating that cholesterol level in the plasma membrane of CT43 is decreased. In addition, the level of cholesterol in the detergent-insoluble, low-density membrane (LDM) fraction of CT43 cells was markedly reduced compared with the other two types of CHO cells. As LDM domain plays critical role in signaling pathways, these results suggest that the reduced cholesterol level in LDM domain due to the lack of NPC1 may activate MAPK, which subsequently promotes tau phosphorylation in NPC1-deficient cells.

Recently, we have found that alterations in cellular cholesterol metabolism are involved in promotion of tau phosphorylation (Fan et al. [2001] J. Neurochem. 76: 391-400; Sawamura et al. [2001] J. Biol. Chem. 276:10314-10319). In addition, we have shown that amyloid beta-protein (Abeta) promotes cholesterol release to form Abeta-lipid particles (Michikawa et al. [2001] J. Neurosci. 21:7226-7235). These lines of evidence inspired us to conduct further studies on whether Abeta affects cholesterol metabolism in neurons, which might lead to tau phosphorylation. Here, we report the effect of Abeta1-40 on cholesterol metabolism in cultured neurons prepared from rat cerebral cortex. Oligomeric Abeta1-40 inhibited cholesterol synthesis and reduced cellular cholesterol levels in a dose- and time-dependent manner, while freshly dissolved Abeta had no effect on cholesterol metabolism. However, oligomeric Abeta had no effect on the proteolysis of sterol regulatory element binding protein-2 (SREBP-2) or protein synthesis in cultured neurons. Oligomeric Abeta did not enhance lactate dehydrogenase (LDH) release from neuronal cells or decrease signals in the cultures reactive to 3,3'-Bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein, hexaacetoxymethyl ester (calcein AM) staining, indicating that Abeta used in this experiment did not cause neuronal death during the time course of our experiments. Since alterations in cholesterol metabolism induce tau phosphorylation, our findings that oligomeric Abeta alters cellular cholesterol homeostasis may provide new insight into the mechanism underlying the amyloid cascade hypothesis. (C) 2002 Wiley-Liss, Inc.

We have reported previously (Michikawa, M., Fan, Q.-W., Isobe, I., and Yanagisawa, K. (2000) J. Neurochem. 74, 1008-1016) that exogenously added recombinant human apolipoprotein E (apoE) promotes cholesterol release in an isoform-dependent manner. However, the molecular mechanism underlying this isoform-dependent promotion of cholesterol release remains undetermined. In this study, we demonstrate that the cholesterol release is mediated by endogenously synthesized and secreted apoE isoforms and clarify the mechanism underlying this apoE isoform-dependent cholesterol release using cultured astrocytes prepared from human apoE3 and apoE4 knock-in mice. Cholesterol and phospholipids were released into the culture media, resulting in the generation of two types of high density lipoprotein (HDL)-like particles; one was associated with apoE and the other with apoJ. The amount of cholesterol released into the culture media from the apoE3-expressing astrocytes was similar to2.5-fold greater than that from apoE4-expressing astrocytes. In contrast, the amount of apoE3 released in association with the HDL-like particles was similar to that of apoE4, and the sizes of the HDL-like particles released from apoE3- and apoE4-expressing astrocytes were similar. The molar ratios of cholesterol to apoE in the HDL fraction of the culture media of apoE3- and apoE4-expressing astrocytes were 250 +/- 6.0 and 119 +/- 5.1, respectively. These data indicate that apoE3 has an ability to generate similarly sized lipid particles with less number of apoE molecules, than apoE4, suggesting that apoE3-expressing astrocytes can supply more cholesterol to neurons than apoE4-expressing astrocytes. These findings provide a new insight into the issue concerning the putative alteration of apoE-related cholesterol metabolism in Alzheimer's disease.

Interactions between amyloid beta -protein (A beta) and lipids have been suggested to play important roles in the pathogenesis of Alzheimer's disease. However, the molecular mechanism underlying these interactions has not been fully understood. We examined the effect of A beta on lipid metabolism in cultured neurons and astrocytes and found that oligomeric A beta, but not monomeric or fibrillar A beta, promoted lipid release from both types of cells in a dose- and time-dependent manner. The main components of lipids released after the addition of A beta were cholesterol, phospholipids, and monosialoganglioside (GM1). Density-gradient and electron microscopic analyses of the conditioned media demonstrated that these A beta and lipids formed particles and were recovered from the fractions at densities of similar to1.08-1.18 g/ml, which were similar to those of high-density lipoprotein (HDL) generated by apolipoproteins. The lipid release mediated by A beta was abolished by concomitant treatment with Congo red and the PKC inhibitor, H7, whereas it was not inhibited with N-acetyl-L-cysteine. These A beta -lipid particles were not internalized into neurons, whereas HDL-like particles produced by apolipoprotein E were internalized. Our findings indicate that oligomeric A beta promotes lipid release from neuronal membrane, which may lead to the disruption of neuronal lipid homeostasis and the loss of neuronal function.

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC, Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3 beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.

The N141I mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease in the Volga German families. We previously reported that mouse brains harboring mutant PS2 contained increased levels of amyloid beta protein (A beta) 42 in the Tris-saline-soluble fraction (Oyama, F., Sawamura, N., Kobayashi, K., Morishima-Kawashima, M., Kuramochi, T., Ito, M., Tomita, T., Maruyama, K., Saido, T. C., Iwatsubo, T., Capell, A., Waiter, J., Grunberg, J., Ueyama, Y., Haass, C. and Ihara, Y. (1998) J. Neurochem. 71, 313-322). Here, using a new extraction protocol, we quantitated the A beta 40 and A beta 42 levels in the Tris-saline-insoluble fraction. The insoluble A beta levels were found to be higher than the soluble A beta levels, and the insoluble A beta 42 levels were markedly increased in mutant PS2 transgenic mice. To investigate the origin of the insoluble A beta 42, we prepared the detergent-insoluble, low density membrane fraction. This fraction from two independent lines of mutant PS2 transgenic mice contained remarkably increased levels of A beta 42 and significantly low levels of glycerophospholipids and sphingomyelin. This unexpected finding suggests that a large increase in the levels of A beta 42 in mutant PS2 mice is presumably induced through alterations of the lipid composition in the low density membrane domain in the brain.

The N141I missense mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease (AD) in the Volga German families. We have generated transgenic mouse lines overexpressing human wild-type or mutant PS2 under transcriptional control of the chicken beta-actin promoter. In the brains of transgenic mice, the levels of human PS2 mRNA were found to be five-to 15-fold higher than that of endogenous mouse PS2 mRNA, The amyloid beta-protein (AP) 42 levels in the brains of mutant PS2 transgenic mice were higher than those in wild-type PS2 transgenic mice at the age of 2, 5, or 8 months. In addition, the A beta 42 levels appeared to increase steadily in the mutant PS2 transgenic mouse brains from 2 to 8 months of age, whereas there was only a small increase in wild-type transgenic mice between the ages of 5 and 8 months. There was no definite difference in the levels of N-terminal and C-terminal fragments between wild-type and mutant PS2 transgenic mice at the age of 2, 5, or 8 months. These data show a definite effect of the PS2 mutation on an age-dependent increase of A beta 42 content in the brain.

To clarify the immunohistochemical features of canine senile plaques (SPs) and cerebral amyloid angiopathy (CAA), the distribution of the amyloid beta protein (A beta) subtypes A beta 40 and A beta 42(43), A beta precursor protein (APP), and glial cell reaction were examined in the brains of seven aged dogs (12-18 years). A beta 42(43) was found to be deposited in all types of SPs, whereas A beta 40 was deposited only in mature (classical and primitive) plaques. CAA, which was located along parenchymal and meningeal arterioles and capillaries, consisted of both subtypes of A beta. APP was exhibited in normal and degenerative neurons and swollen neurites of mature plaques. It was, therefore, considered that A beta 42(43) in diffuse plaques might be derived from APP in neurons, while A beta 40 and A beta 42(43) in mature plaques might be generated from APP in swollen neurites in the plaque. In contrast to the case in humans, in whom deposition of A beta 40 and A beta 42(43) in the mature plaques is predominantly associated with microglial reaction, in dogs we found that it was closely associated with astroglial reaction. The present findings showed characteristics of canine SPs which are different from those of humans.

We analyzed the composition of amyloid beta protein (A beta) species in cerebral amyloid angiopathy (CAA) of an aged squirrel monkey. Immunocytochemistry demonstrated that the cerebral cortex contained no lesions other than widespread CAA with A beta 40 as its apparent major component. However, enzyme-linked immunosorbent assay revealed that A beta 42(43) predominated over A beta 40 in a formic acid-extracted cortical fraction. These findings suggest possible underestimation of A beta 42(43) levels in some previous immunocytochemical investigations. (C) 1997 Elsevier Science B.V.

To investigate the pathomechanism of amyloid beta protein (A beta) deposition in brains with Alzheimer's disease (AD), cerebrospinal fluid (CSF) levels of A beta species (CSF-A beta) with different carboxy termini, i.e. A beta X-40 and A beta X-42(43) as well as A beta 1-40 and A beta 1-42(43), were measured in patients with AD and age-matched controls without dementia (CTR) using sandwich enzyme-linked immunosorbent assays (ELISAs). The present study revealed that both CSF-A beta X-42(43) and A beta 1-42(43) levels were significantly lower in the AD patients (P<0.005) than in the CTR group, whereas neither CSF-A beta X-40 nor CSF-A beta 1-40 levels showed any differences between the two groups. In addition, although there was no difference between the ratios of A beta X-40 to A beta 1-40 in the AD and CTR groups, the ratios of A beta X-42(43) to A beta 1-42(43) were increased in the AD group compared with those in the CTR group (P<0.05). Therefore, it can be assumed that the ratios of amino terminal truncations and/or modifications of CSF-A beta 42(43) with carboxy termini ending at residue 42(43) were more increased in the AD group than in the CTR group. Increased adsorption of A beta 42(43) to A beta deposition in AD brains, decreased secretion of A beta 42(43) to CSF and/or increased clearance of A beta 42(43) from CSF might explain the diminished levels of A beta 42(43) in the CSF of AD patients. In addition, CSF-A beta 42(43) could reflect increased amino terminal truncations and/or modifications of A beta 42(43) in AD brains. (C) 1997 Elsevier Science B.V.

Fibrillar amyloid beta protein (A beta) deposition is increased in the brains of patients with Alzheimer's disease (AD), and is manifested as senile plaques (SPs) and congophilic angiopathy (CA). A beta 40 and A beta 42(43), two chief species of A beta, are documented in SPs and CA, as well as in cerebrospinal fluid (CSF) and cell culture media. A beta 42(43) is the major component of diffuse plaques, the earliest form of SPs. Thus, we hypothesized that determination of the amount of A beta 42(43) in CSF or plasma might provide a diagnostic laboratory test for AD. We measured amounts of different A beta species in plasma from 28 patients with sporadic probable AD, 40 age-matched neurologic patients without dementia and 25 age-matched normal controls using enzyme-linked immunosorbent assays (ELISAs). Plasma concentrations of A beta 1-40 and A beta 1-42(43) did not significantly differ among these groups. These findings suggest the unlikelihood that plasma A beta assays would be useful as a diagnostic tool for AD.

Senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in the brains of five aged (20-26 years old) cynomolgus monkeys were investigated immunohistochemically using two monoclonal antibodies (anti-A beta 40 (BA27) and anti-A beta 42(43) (BC05)) that can differentiate the carboxyl termini of amyloid beta protein (A beta) subtypes. In four of five animals, all types of SPs (i.e. diffuse, primitive, and classical plaques; DPs, PPs, and CPs, respectively) were identified by BC05. However, BA27 did not label DPs and stained only about one third of PPs and CPs, mainly labeling granular structures and cored portions, respectively. In CAA, lesions of cortical capillaries reacted to BC05 in four of five cases, but rarely and weakly to BA27 in two of five cases. On the other hand, lesions of parenchymal and meningeal arterioles were stained by both BA27 and BC05. These staining profiles of SPs in cynomolgus monkeys correspond well to those in humans, although there are two remarkable features in cynomolgus monkeys. First, BA27 stained PPs associated with granular structures. Secondly, capillary A beta reacted intensely to BC05 but only slightly to BA27. Despite these unique features, the results suggest that aged cynomolgus monkeys can be used to investigate the pathogenesis of A beta deposition in SPs and CAA.

Diffuse plaques are immature and amorphous senile plaques and believed to be in the initial phase of plaque formation. In contrast to amyloid angiopathy and the plaque core amyloid, diffuse plaques failed to be purified in preserved forms from the brain. Here, we studied the diffuse plaques in the cerebellar region of the Alzheimer's disease brain based on immunocytochemistry and ELISA using two different monoclonal antibodies specifically recognizing the carboxyl termini of A beta molecules (BA27 for A beta 1-40 and BC05 for A beta 1-42/43). We found that the amount of A beta 1-40 was in proportion to the staining degree on amyloid angiopathy by immunohistochemistry. We found that A beta 1-42/43 comprised diffuse plaques as the major component in the cerebella of AD brains. Taking these findings into consideration, diffuse plaques, the earliest pathological change in the brain with AD, are concluded to be composed mainly of A beta 1-42/43, implicating the critical importance of this kind of A beta species deposition in the pathogenesis of AD.

We measured the amounts of total A beta, A beta 1-40, and A beta 1-42/43 in brain tissues using a newly developed ELISA assay and found that the amounts of insoluble A beta 1-42/43 and insoluble A beta 1-40) were linearly related to the amount of A beta deposits or total insoluble A beta at their lon er and higher concentrations, respectively. In an experiment to characterize the A beta species in brain homogenates with buffered saline, we unexpectedly detected soluble AB which was derived from the insoluble amyloid deposits in brain tissue, indicating reversible depolymerisation of AR from insoluble amyloid deposits. To confirm this finding, we performed 5 consecutive washes of insoluble precipitates of AD brains with buffered saline. Bath species of A beta were found in all 5 supernatant fractions and their amounts were gradually decreased. The ratio of A beta 1-42/43/43 to A beta-40 was increased with the numbers of washes, indicating that A beta 1-42/43) existed in an exposed manner as compared to A beta 1-42/43. Thus the present finding is the first biochemical evidence that A beta 1-40, was the predominant species involved in the reversible exchanging reaction on seeding A beta 1-42/43 between the soluble and the insoluble forms (amyloid fibrils). (C) 1994 Academic Press, Inc.

膜貫通型タンパク質であるニューレグリン(NRG)1の切断異常により、統合失調症をはじめとする精神神経疾患発症に至るという仮説のもとに、分子生物学生化学的手法によりNRG1の切断機構を解明することにより、統合失調症をはじめとする精神神経疾患の創薬ターゲットの同定を目指して研究した。NRG1の断片を認識する抗体を用いて免疫沈降法を行い、NRG断片の濃縮精製を試みた。NRG1を発現した大量の細胞のライセートに抗体とプロテインAセファロースを混ぜて反応させた。熱処理によりサンプルを回収し、電気泳動を行い、ゲルから目的の断片を回収した。NRG1タンパクの断片を抗NRG1抗体による免疫沈降法により精製することに成功した。