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Increased oxidative damage is a prominent and early feature in Alzheimer disease. We previously crossed Alzheimer disease transgenic (APPsw) model mice with alpha-tocopherol transfer protein knock-out (Ttpa(-/-)) mice in which lipid peroxidation in the brain was significantly increased. The resulting double-mutant (Ttpa(-/-)APPsw) mice showed increased amyloid beta (Abeta) deposits in the brain, which was ameliorated with alpha-tocopherol supplementation. To investigate the mechanism of the increased Abeta accumulation, we here studied generation, degradation, aggregation, and efflux of Abeta in the mice. The clearance of intracerebral-microinjected (125)I-Abeta(1-40) from brain was decreased in Ttpa(-/-) mice to be compared with wild-type mice, whereas the generation of Abeta was not increased in Ttpa(-/-)APPsw mice. The activity of an Abeta-degrading enzyme, neprilysin, did not decrease, but the expression level of insulin-degrading enzyme was markedly decreased in Ttpa(-/-) mouse brain. In contrast, Abeta aggregation was accelerated in Ttpa(-/-) mouse brains compared with wild-type brains, and well known molecules involved in Abeta transport from brain to blood, low density lipoprotein receptor-related protein-1 (LRP-1) and p-glycoprotein, were up-regulated in the small vascular fraction of Ttpa(-/-) mouse brains. Moreover, the disappearance of intravenously administered (125)I-Abeta(1-40) was decreased in Ttpa(-/-) mice with reduced translocation of LRP-1 in the hepatocytes. These results suggest that lipid peroxidation due to depletion of alpha-tocopherol impairs Abeta clearances from the brain and from the blood, possibly causing increased Abeta accumulation in Ttpa(-/-)APPsw mouse brain and plasma.

The mechanism underlying plaque-independent neuronal death in Alzheimer disease (AD), which is probably responsible for early cognitive decline in AD patients, remains unclarified. Here, we show that a toxic soluble Abeta assembly (TAbeta) is formed in the presence of liposomes containing GM1 ganglioside more rapidly and to a greater extent from a hereditary variant-type ("Arctic") Abeta than from wild-type Abeta. TAbeta is also formed from soluble Abeta through incubation with natural neuronal membranes prepared from aged mouse brains in a GM1 ganglioside-dependent manner. An oligomer-specific antibody (anti-Oligo) significantly suppresses TAbeta toxicity. Biophysical and structural analyses by atomic force microscopy and size exclusion chromatography revealed that TAbeta is spherical with diameters of 10-20 nm and molecular masses of 200-300 kDa. TAbeta induces neuronal death, which is abrogated by the small interfering RNA-mediated knockdown of nerve growth factor receptors, including TrkA and p75 neurotrophin receptor. Our results suggest that soluble Abeta assemblies, such as TAbeta, can cause plaque-independent neuronal death that favorably occurs in nerve growth factor-dependent neurons in the cholinergic basal forebrain in AD.

The acquisition of neuronal type-specific morphogenesis is a central feature of neuronal differentiation and has important consequences for region-specific nervous system functions. Here, we report that the cell type-specific cholesterol profile determines the differential modulation of axon and dendrite outgrowths in hippocampal and cerebral cortical neurons in culture. The extent of axon and dendrite outgrowths is greater and the polarity formation occurs earlier in cortical neurons than in hippocampal neurons. The cholesterol concentrations in total homogenate and the lipid rafts from hippocampal neurons are significantly higher than those from cortical neurons. Cholesterol depletion by beta-cyclodextrin markedly enhanced the neurite outgrowth and accelerated the establishment of neuronal polarity in hippocampal neurons, which were similarly observed in nontreated cortical neurons, whereas cholesterol loading had no effects. In contrast, both depletion and loading of cholesterol decreased the neurite outgrowths in cortical neurons. The stimulation of neurite outgrowth and polarity formation induced by cholesterol depletion was accompanied by an enhanced localization of Fyn, a Src kinase, in the lipid rafts of hippocampal neurons. A concomitant treatment with beta-cyclodextrin and a Src family kinase inhibitor, PP2, specifically blocked axon outgrowth but not dendrite outgrowth (both of which were enhanced by beta-cyclodextrin) in hippocampal neurons, suggesting that axon outgrowth modulated by cholesterol is induced in a Fyn-dependent manner. These results suggest that cellular cholesterol modulates axon and dendrite outgrowths and neuronal polarization under culture conditions and also that the difference in cholesterol profile between hippocampal and cortical neurons underlies the difference in neurite outgrowth between these two types of neurons.

Niemann-Pick type C1 (NPC1) disease is an autosomal recessive, fatal disorder characterized by a defect in cholesterol trafficking and progressive neurodegeneration. The disease is predominantly caused by mutations in the NPC1 gene; however, it has been assumed that heterozygous NPC1 mutations do not cause any symptoms. Here we demonstrate that cholesterol accumulation does not occur in young mouse brains; however, it does in aged (104-106-week-old) NPC1+/- mouse brains. In addition, Purkinje cell loss was observed in aged NPC1+/- mouse cerebellums. Immunoblot analysis using anti-phospho-tau antibodies (AT-8, AT-100, AT-180, AT-270, PHF-1, and SMI-31) demonstrates the site-specific phosphorylation of tau at Ser-199, Ser-202, Ser-212, and Thr-214 in the brains of aged NPC1+/- mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases, including glycogen synthase kinase 3beta, cyclin-dependent kinase 5, or stress-activated protein kinase/c-Jun N-terminal kinase were not activated. Cholesterol level in the lipid raft isolated from the cerebral cortices, ATP level, and ATP synthase activity in the cerebral cortices significantly decreased in the aged NPC1+/- brains compared with those in the NPC1+/+ brains. All of these changes observed in NPC1+/- brains were determined to be associated with aging and were not observed in the age-matched NPC1+/+ brains. These results clearly demonstrate that heterozygous NPC1 impairs neuronal functions and causes neurodegeneration in aged mouse brains, suggesting that human heterozygous NPC1 mutations may be a risk factor for neurodegenerative disorders, such as tauopathy, in the aged population.

Familial Alzheimer disease-causing mutations in the presenilins increase production of longer pathogenic amyloid beta-peptides (A beta(42/43)) by altering gamma-secretase activity. The mechanism underlying this effect remains unknown, although it has been proposed that heteromeric macromolecular complexes containing presenilins mediate gamma-secretase cleavage of the amyloid beta-precursor protein. Using a random mutagenesis screen of presenilin-1 (PS1) for PS1 endoproteolysis-impairing mutations, we identified five unique mutants, including R278I-PS1 and L435H-PS1, that exclusively generated a high level of A beta43, but did not support physiological PS1 endoproteolysis or A beta40 generation. These mutants did not measurably alter the molecular size or subcellular localization of PS1 complexes. Pharmacological studies indicated that the up-regulation of activity for A beta43 generation by these mutations was not further enhanced by the difluoroketone inhibitor DFK167 and was refractory to inhibition by sulindac sulfide. These results suggest that PS1 mutations can lead to a wide spectrum of changes in the activity and specificity of gamma-secretase and that the effects of PS1 mutations and gamma-secretase inhibitors on the specificity are mediated through a common mechanism.

Niemann-Pick type C1 (NPC1) disease is a fatal hereditary disorder characterized by a defect in cholesterol trafficking and progressive neurodegeneration. Although the NPC1 gene has been identified, the molecular mechanism responsible for neuronal dysfunction in brains of patients with NPC1 disease remains unknown. This study demonstrates that the amount of cholesterol within mitochondria membranes is significantly elevated in NPC1 mouse brains and neural cells. In addition, the mitochondrial membrane potential, the activity of ATP synthase, and henceforth the level of ATP are markedly decreased in NPC1 mouse brains and neurons. Importantly, reducing the level of cholesterol within mitochondrial membranes using methyl-beta-cyclodextrin can restore the activity of ATP synthase. Finally, NPC1 neurons show an impaired neurite outgrowth, which can be rescued by exogenous ATP. These results suggest that mitochondrial dysfunctions and subsequent ATP deficiency, which are induced by altered cholesterol metabolism in mitochondria, may be responsible for neuronal impairment in NPC1 disease.

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 beta-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.

Presenilin (PS) is essential for gamma-cleavage, which is required for the generation of amyloid beta-protein (Abeta) from the beta-amyloid precursor protein. However, it remains to be clarified how gamma-cleavage is regulated. To elucidate the regulation of PS-mediated gamma-cleavage, we developed a new functional screening method for identifying cDNA that enhances gamma-cleavage. This screening system utilizes our own developed cell line, where the expression of cDNA that enhances gamma-cleavage confers puromycin resistance. The cDNA library is retrovirally delivered to the above-mentioned cell line, allowing the identification of our target cDNAs by a combination of puromycin resistance selection and Abeta assay screening. With this screening method, we isolated several cDNAs enhancing gamma-cleavage, including the previously reported Herp. Here we also demonstrate that Rab1A, identified with this screening, can be a regulator of Abeta generation. Thus, our established screening method is a powerful tool for identifying multiple regulators involved in gamma-cleavage in the Abeta generation pathway, including modulators of gamma-secretase activity or the intracellular trafficking of factors necessary for gamma-cleavage.

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 approximately 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 Alzheimer's disease.

Presenilin (PS) is essential for the gamma-cleavage required for the generation of the C terminus of amyloid beta-protein (Abeta). However, the mechanism underlying PS-mediated gamma-cleavage remains unclear. We have identified Herp cDNA by our newly developed screening method for the isolation of cDNAs that increase the degree of gamma-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 Abeta generation, although not in PS-deficient cells. We found that Herp interacts with both PS1 and PS2. Thus, Herp regulates PS-mediated Abeta 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 Abeta accumulation, including the formation of senile plaques and vascular Abeta deposits.

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-3beta 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.