Katsuhiko Yanagisawa

A Seed for Alzheimer Amyloid in the Brain

A fundamental question about the early pathogenesis of Alzheimers disease (AD) concerns how toxic aggregates of amyloid β protein (Aβ) are formed from its nontoxic soluble form. We hypothesized previously that GM1 ganglioside-bound Aβ (GAβ) is involved in the process. We now examined this possibility using a novel monoclonal antibody raised against GAβ purified from an AD brain. Here, we report that GAβ has a conformation distinct from that of soluble Aβ and initiates Aβ aggregation by acting as a seed. Furthermore, GAβ generation in the brain was validated by both immunohistochemical and immunoprecipitation studies. These results imply a mechanism underlying the onset of AD and suggest that an endogenous seed can be a target of therapeutic strategy.

Apolipoprotein E Exhibits Isoform‐Specific Promotion of Lipid Efflux from Astrocytes and Neurons in Culture

Abstract: Many studies have shown that apolipoprotein E (apoE) plays important roles in maintaining intracellular lipid homeostasis in nonneuronal cells. However, little is known about the extracellular transport of lipids in the CNS. In this study, we determined whether and to what degree lipid efflux from astrocytes and neurons depended on apoE. Our results showed that exogenously added apoE promoted the efflux of cholesterol and phosphatidylcholine from both astrocytes and neurons in culture, resulting in the generation of high‐density lipoprotein‐like particles. The order of potency of the apoE isoforms as lipid acceptors was apoE2 > apoE3 = apoE4 in astrocytes and apoE2 > apoE3 > apoE4 in neurons. Treatment with brefeldin A, monensin, and a protein kinase C inhibitor, H7, abolished the ability of apoE to promote cholesterol efflux from cultured astrocytes, without altering apoE‐mediated phosphatidylcholine efflux. In contrast, the efflux of both cholesterol and phosphatidylcholine promoted by apoE was abolished following treatment with heparinase or lactoferrin, which block the interaction of apoE with heparan sulfate proteoglycans (HSPGs) or low‐density lipoprotein receptor‐related protein (LRP), respectively. This study suggests that apoE promotes lipid efflux from astrocytes and neurons in an isoform‐specific manner and that cell surface HSPGs and/or HSPG‐LRP pathway may mediate this apoE‐promoted lipid efflux.

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.

Aβ polymerization through interaction with membrane gangliosides

Clarification of the molecular and cellular mechanisms underlying the assembly of amyloid beta-protein (Abeta) into insoluble fibrils in the brain has been one of the biggest challenges in the research on Alzheimer disease (AD). We previously identified a novel Abeta species, which was characterized by its tight binding to GM1 ganglioside (GM1), in the brain showing early pathological changes of AD. The ganglioside-bound Abeta (GAbeta) possessed unique characteristics, including its altered immunoreactivity, which suggests its distinct conformation from native Abeta, and its strong potency to accelerate Abeta assembly into fibrils. On the basis of these characteristics, it was hypothesized that Abeta adopts an altered conformation following interaction with GM1, leading to the generation of GAbeta, and then GAbeta acts as an endogenous seed for Alzheimer amyloid in the brain. To date, various in vitro and in vivo studies on GAbeta have revealed how Abeta binds to gangliosides, i.e., what are the favorable physicochemical and neurobiological conditions for generating GAbeta, and what is the pathological significance of ganglioside-induced Abeta assembly in the development of AD. Interestingly, GAbeta is favorably generated in the unique ganglioside-enriched (clustered), raft-like microdomains; moreover, amyloid fibrils formed in the presence of gangliosides are neurotoxic. Furthermore, the conformational change of Abeta in the presence of ganglioside has been characterized by an NMR study. In this review, we focus on the recent progress of GAbeta studies and highlight the possibility that ganglioside binding is the initial and common step in the development of a part of human misfolding-type amyloidoses, including AD.

Inhibition of Cholesterol Production but Not of Nonsterol Isoprenoid Products Induces Neuronal Cell Death

Abstract: Deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, has been known to cause an inhibition of DNA synthesis and cell growth, and to induce apoptosis in nonneuronal cells. To investigate whether this is also the case in neurons, we examined the effect of a 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor on the viability of neuronal cultures prepared from fetal rat brains. Treatment with compactin, a competitive inhibitor of HMG‐CoA reductase, induced neuronal death in a dose‐dependent manner. Concurrent treatment with cholesterol, β‐migrating very low density lipoprotein, mevalonate, or squalene substantially inhibited the induction of neuronal death by compactin. Cell death was also induced by treatment with squalestatin, which specifically inhibits cholesterol biosynthesis at a site down‐stream from the generation of nonsterol metabolites. Furthermore, squalestatin‐induced neuronal death was inhibited by concurrent incubation with squalene but not mevalonate. In contrast, cell growth of proliferating cells such as NIH 3T3 and PC12 cells was exclusively dependent on the level of nonsterol isoprenoid products and not that of cholesterol. The results of this study clearly indicate that the viability of neurons, different from that of nonneuronal cells, depends on the intracellular cholesterol content and not on the intermediate nonsterol isoprenoid products.

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 tau phosphorylation in cultured neurons

One of the hallmarks of Alzheimers disease (AD) is the abnormal state of tau. It is both highly phosphorylated and aggregated into paired helical filaments (PHFs) in neurofibrillary tangles (NFTs). However, the mechanism underlying the hyperphosphorylation of tau in NFTs and neuronal degeneration in AD remains to be elucidated. The fact that hyperphosphorylation of tau in NFTs are also found in the patients with Niemann–Pick disease, type C (NPC), which is a cholesterol storage disease associated with defective intracellular trafficking of exogenous cholesterol, implies that perturbation of cholesterol metabolism may be involved in tau phosphorylation and neurodegeneration. Here, we report that cholesterol deficiency induced by inhibition of cholesterol biosynthesis in cultured neurons results in hyperphosphorylation of tau, accompanied by axonal degeneration associated with microtubule depolymerization. These changes were prevented by concurrent treatment with β‐migrating very low‐density lipoprotein (β‐VLDL) or cholesterol. We propose that intracellular cholesterol plays an essential role in the modulation of tau phosphorylation and the maintenance of microtubule stability.

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.

Endoplasmic reticulum stress-inducible protein, Herp, enhances presenilin-mediated generation of amyloid beta-protein

Presenilin (PS) is essential for the γ-cleavage required for the generation of the C terminus of amyloid β-protein (Aβ). However, the mechanism underlying PS-mediated γ-cleavage remains unclear. We have identified Herp cDNA by our newly developed screening method for the isolation of cDNAs that increase the degree of γ-cleavage. Herp was originally identified as a homocysteine-responsive protein, and its expression is up-regulated by endoplasmic reticulum stress. Herp is an endoplasmic reticulum-localized membrane protein that has a ubiquitin-like domain. Here, we report that a high expression of Herp in cells increases the level of Aβ generation, although not in PS-deficient cells. We found that Herp interacts with both PS1 and PS2. Thus, Herp regulates PS-mediated Aβ generation, possibly through its binding to PS. Immunohistochemical analysis of a normal human brain section with an anti-Herp antibody revealed the exclusive staining of neurons and vascular smooth muscle cells. Moreover, the antibody strongly stained activated microglia in senile plaques in the brain of patients with Alzheimer disease. Taken together, Herp could be involved in Aβ accumulation, including the formation of senile plaques and vascular Aβ deposits.

Age-Dependent Change in the Levels of Aβ40 and Aβ42 in Cerebrospinal Fluid from Control Subjects, and a Decrease in the Ratio of Aβ42 to Aβ40 Level in Cerebrospinal Fluid from Alzheimer’s Disease Patients

In order to address an age-dependent alteration in the concentration of β-amyloid polypeptides (Aβs) within the central nervous system and its probable predisposition to amyloidgenesis in Alzheimer’s disease (AD), we measured two species of soluble Aβs, Aβ40 and Aβ42, in cerebrospinal fluids (CSF) from randomly selected Japanese control subjects at various ages (n = 33) and then compared these data with those of probable Japanese AD patients (n = 23). CSF concentrations of Aβ40 and Aβ42 peptides were age-dependent (ANOVA, Bonferroni’s multiple comparison; p < 0.01 and p < 0.05, respectively) and were lower in the infant than in adults. From mid-20, the Aβ40 concentrations were decreasing while Aβ42 were rather stable. Aβs in CSF from AD patients (n = 23), whose ε4 allele frequency of the apolipoprotein E gene was higher than in controls (n = 83, p < 0.03), were not statistically different from those of age-matched controls (n = 13). A linear relationship was detected between the Aβ40 concentration and the Mini-Mental State Examination score (p < 0.05). The ratio of the Aβ42 to the Aβ40 level measured in the AD CSF samples was approximately 38% decreased compared to age-matched controls (p < 0.05). These data suggest that the physiological metabolism of soluble Aβs in the brain is regulated in an age-dependent manner, and that the ratio of Aβ42 to Aβ40 level in the CSF would be a useful marker for monitoring progression of AD.