Naoya Sawamura

A schizophrenia-associated mutation of DISC1 perturbs cerebral cortex development

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 wild-type 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 DISC1 function may underlie neurodevelopmental dysfunction in schizophrenia.

Apolipoprotein E (ApoE) isoform-dependent lipid release from astrocytes prepared from human ApoE3 and ApoE4 knock-in mice.

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 ∼2.5-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 Alzheimers disease.

Amyloid β-protein (Aβ)1–40 protects neurons from damage induced by Aβ1–42 in culture and in rat brain

Previously, we found that amyloid β‐protein (Aβ)1–42 exhibits neurotoxicity, while Aβ1–40 serves as an antioxidant molecule by quenching metal ions and inhibiting metal‐mediated oxygen radical generation. Here, we show another neuroprotective action of nonamyloidogenic Aβ1–40 against Aβ1–42‐induced neurotoxicity in culture and in vivo. Neuronal death was induced by Aβ1–42 at concentrations higher than 2 μm, which was prevented by concurrent treatment with Aβ1–40 in a dose‐dependent manner. However, metal chelators did not prevent Aβ1–42‐induced neuronal death. Circular dichroism spectroscopy showed that Aβ1–40 inhibited the β‐sheet transformation of Aβ1–42. Thioflavin‐T assay and electron microscopy analysis revealed that Aβ1–40 inhibited the fibril formation of Aβ1–42. In contrast, Aβ1–16, Aβ25–35, and Aβ40–1 did not inhibit the fibril formation of Aβ1–42 nor prevent Aβ1–42‐induced neuronal death. Aβ1–42 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 Aβ1–40. These results indicate that Aβ1–40 protects neurons from Aβ1–42‐induced neuronal damage in vitro and in vivo, not by sequestrating metals, but by inhibiting the β‐sheet transformation and fibril formation of Aβ1–42. Our data suggest a mechanism by which elevated Aβ1–42/Aβ1–40 ratio accelerates the development of Alzheimers disease (AD) in familial AD.

Cholesterol-dependent modulation of dendrite outgrowth and microtubule stability in cultured neurons

Microtubule‐associated protein 2 (MAP2) is a neuron‐specific cytoskeletal protein enriched in dendrites and cell bodies. MAP2 regulates microtubule stability in a phosphorylation‐dependent manner, which has been implicated in dendrite outgrowth and branching. We have previously reported that cholesterol deficiency causes tau phosphorylation and microtubule depolymerization in axons ( Fan et al. 2001 ). To investigate whether cholesterol also modulates microtubule stability in dendrites by modulating MAP2 phosphorylation, we examined the effect of compactin, a 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor, and TU‐2078 (TU), a squalene epoxidase inhibitor, on these parameters using cultured neurons. We have found that cholesterol deficiency induced by compactin and TU, inhibited dendrite outgrowth, but not of axons, and attenuated axonal branching. Dephosphorylation of MAP2 and microtubule depolymerization accompanied these alterations. The amount of protein phosphatase 2 A (PP2A) and its activity in association with microtubules were decreased, while those unbound to microtubules were increased. The synthesized ceramide levels and the total ceramide content were increased in these cholesterol‐deficient neurons. These alterations caused by compactin were prevented by concurrent treatment of cultured neurons with β‐migrating very‐low‐density lipoproteins (β‐VLDL) or cholesterol. Taken together, we propose that cholesterol‐deficiency causes a selective inhibition of dendrite outgrowth due to the decreased stability of microtubules as a result of inhibition of MAP2 phosphorylation.

Mutant Presenilin 2 Transgenic Mouse: Effect on an Age-Dependent Increase of Amyloid β-Protein 42 in the Brain†

Abstract: The N141I missense mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimers 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 β‐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 β‐protein (Aβ) 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 Aβ42 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 Aβ42 content in the brain.

Nuclear DISC1 regulates CRE-mediated gene transcription and sleep homeostasis in the fruit fly

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.

Amyloid β-protein affects cholesterol metabolism in cultured neurons: Implications for pivotal role of cholesterol in the amyloid cascade

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 β‐protein (Aβ) promotes cholesterol release to form Aβ‐lipid particles (Michikawa et al. [ 2001 ] J. Neurosci. 21:7226–7235). These lines of evidence inspired us to conduct further studies on whether Aβ affects cholesterol metabolism in neurons, which might lead to tau phosphorylation. Here, we report the effect of Aβ1‐40 on cholesterol metabolism in cultured neurons prepared from rat cerebral cortex. Oligomeric Aβ1‐40 inhibited cholesterol synthesis and reduced cellular cholesterol levels in a dose‐ and time‐dependent manner, while freshly dissolved Aβ had no effect on cholesterol metabolism. However, oligomeric Aβ had no effect on the proteolysis of sterol regulatory element binding protein‐2 (SREBP‐2) or protein synthesis in cultured neurons. Oligomeric Aβ 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 Aβ 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 Aβ alters cellular cholesterol homeostasis may provide new insight into the mechanism underlying the amyloid cascade hypothesis.

Promotion of tau phosphorylation by MAP kinase Erk1/2 is accompanied by reduced cholesterol level in detergent-insoluble membrane fraction in Niemann-Pick C1-deficient cells.

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.

Primate disrupted-in-schizophrenia-1 (DISC1): High divergence of a gene for major mental illnesses in recent evolutionary history

Here we analyze the species conservation of disrupted-in-schizophrenia-1 (DISC1) gene, a susceptibility gene for schizophrenia. We cloned cDNA of DISC1 and characterized DISC1 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 DISC1 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 DISC1 sequences in chicken and Caenorhabditis elegans, but failed to find DISC1 in Drosophila. In contrast to sequence differences, the regional expression profile of DISC1 is well conserved between rodents and primates in that levels of DISC1 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.

Cereblon is recruited to aggresome and shows cytoprotective effect against ubiquitin-proteasome system dysfunction

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.