Wataru Araki

Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation.

Familial Alzheimer disease mutations of presenilin 1 (PS-1) enhance the generation of Aβ1–42, indicating that PS-1 is involved in amyloidogenesis. However, PS-1 transgenic mice have failed to show amyloid plaques in their brains. Because PS-1 mutations facilitate apoptotic neuronal death in vitro, we did careful quantitative studies in PS-1 transgenic mice and found that neurodegeneration was significantly accelerated in mice older than 13 months (aged mice) with familial Alzheimer disease mutant PS-1, without amyloid plaque formation. However, there were significantly more neurons containing intracellularly deposited Aβ42 in aged mutant transgenic mice. Our data indicate that the pathogenic role of the PS-1 mutation is upstream of the amyloid cascade.

Trophic effect of β-amyloid precursor protein on cerebral cortical neurons in culture

We investigated the effect of human beta-amyloid precursor protein (APP) on rat primary cerebral cortical neurons cultured in a serum-free medium. Two secretory APP species (APP667 and APP592) with and without the protease inhibitor domain were produced by COS-1 cells transfected with APP cDNAs, which encode the N-terminal portions of APP770 and APP695. Both highly purified APP species, when added to the medium, enhanced neuronal survival and neurite extension in a dose-dependent manner with a maximum effect at approximately 100 nM. These results suggest that secreted forms of APP have trophic activity for cerebral cortical neurons.

Reticulons RTN3 and RTN4‐B/C interact with BACE1 and inhibit its ability to produce amyloid β‐protein

β‐Secretase β‐site APP cleaving enzyme 1 (BACE1), is a membrane‐bound aspartyl protease necessary for the generation of amyloid β‐protein (Aβ), which accumulates in the brains of individuals with Alzheimers disease (AD). To gain insight into the mechanisms by which BACE1 activity is regulated, we used proteomic methods to search for BACE1‐interacting proteins in human neuroblastoma SH‐SY5Y cells, which overexpress BACE1. We identified reticulon 4‐B (RTN4‐B; Nogo‐B) as a BACE1‐associated membrane protein. Co‐immunoprecipitation experiments confirmed a physical association between BACE1 and RTN4‐B, RTN4‐C (the shortest isoform of RTN‐4), and their homologue reticulon 3 (RTN3), both in SH‐SY5Y cells and in transfected human embryonic kidney (HEK) 293 cells. Overexpression of these reticulons (RTNs) resulted in a 30–50% reduction in the secretion of both Aβ40 and Aβ42 from HEK293 cells expressing the AD‐associated Swedish mutant amyloid precursor protein (APP), but did not affect Aβ secretion from cells expressing the APP β‐C‐terminal fragment (β‐CTF), indicating that these RTNs can inhibit BACE1 activity. Furthermore, a BACE1 mutant lacking most of the N‐terminal ectodomain also interacted with these RTNs, suggesting that the transmembrane region of BACE1 is critical for the interaction. We also observed a similar interaction between these RTNs and the BACE1 homologue BACE2. Because RTN3 and RTN4‐B/C are substantially expressed in neural tissues, our findings suggest that they play important roles in the regulation of BACE1 function and Aβ production in the brain.

Involvement of Prostaglandin E2 in Production of Amyloid-β Peptides Both in Vitro and in Vivo

Amyloid-β peptides (Aβ), generated by proteolysis of the β-amyloid precursor protein (APP) by β- and γ-secretases, play an important role in the pathogenesis of Alzheimer disease (AD). Inflammation is also believed to be integral to the pathogenesis of AD. Here we show that prostaglandin E2 (PGE2), a strong inducer of inflammation, stimulates the production of Aβ in cultured human embryonic kidney (HEK) 293 or human neuroblastoma (SH-SY5Y) cells, both of which express a mutant type of APP. We have demonstrated using subtype-specific agonists that, of the four main subtypes of PGE2 receptors (EP1–4), EP4 receptors alone or EP2 and EP4 receptors together are responsible for this PGE2-stimulated production of Aβ in HEK293 or SH-SY5Y cells, respectively. An EP4 receptor antagonist suppressed the PGE2-stimulated production of Aβ in HEK293 cells. This stimulation was accompanied by an increase in cellular cAMP levels, and an analogue of cAMP stimulated the production of Aβ, demonstrating that increases in the cellular level of cAMP are responsible for the PGE2-stimulated production of Aβ. Immunoblotting experiments and direct measurement of γ-secretase activity suggested that PGE2-stimulated production of Aβ is mediated by activation ofγ-secretase but not of β-secretase. Transgenic mice expressing the mutant type of APP showed lower levels of Aβ in the brain, when they were crossed with mice lacking either EP2 or EP4 receptors, suggesting that PGE2-mediated activation of EP2 and EP4 receptors is involved in the production of Aβ in vivo and in the pathogenesis of AD.

Enhanced generation of intracellular Aβ42 amyloid peptide by mutation of presenilins PS1 and PS2

The accumulation of amyloid β‐peptide (Aβ) in the brain is a critical pathological process in Alzheimers disease (AD). Recent studies have implicated intracellular Aβ in neurodegeneration in AD. To investigate the generation of intracellular Aβ, we established human neuroblastoma SH‐SY5Y cells stably expressing wild‐type amyloid precursor protein (APP), Swedish mutant APP, APP plus presenilin 1 (PS1) and presenilin 2 (PS2; wild‐type or familial AD‐associated mutant), and quantified intracellular Aβ40 and Aβ42 in formic acid extracts by sensitive Western blotting. Levels of both intracellular Aβ40 and Aβ42 were 2–3‐fold higher in cells expressing Swedish APP, compared with those expressing wild‐type APP. Intracellular Aβ42/Aβ40 ratios were approximately 0.5 in these cells. These ratios were increased markedly in cells expressing mutant PS1 or PS2 compared with those expressing their wild‐type counterparts, consistent with the observed changes in secreted Aβ42/Aβ40 ratios. High total levels of intracellular Aβ were observed in cells expressing mutant PS2 because of a marked elevation of Aβ42. Immunofluorescence staining additionally revealed more intense Aβ42 immunoreactivity in mutant PS2‐expressing cells than in wild‐type cells, which was partially colocalized with immunoreactivity for the trans‐Golgi network and endosomes. The data collectively indicate that PS mutations promote the accumulation of intracellular Aβ42, which appears to be localized in multiple subcellular compartments.

Familial frontotemporal dementia and parkinsonism with a novel mutation at an intron 10+11-splice site in the tau gene

We report a case of familial frontotemporal dementia and parkinsonism characterized by early onset with mental retardation. The patient died at the age of 54; neuronal loss was severe in the frontal and temporal cortices, globus pallidus, substantia nigra, red nucleus and dentate nucleus. Anti‐tau‐positive fibrillary changes were observed in neurons and glia in these regions. Although the patient had 2 novel point mutations of the tau gene, P301P (CCG to CCA) and an intron 10+11‐splice site (T to C), exon trapping analysis indicated that the latter was pathogenic.

Homocysteic acid induces intraneuronal accumulation of neurotoxic Aβ42: Implications for the pathogenesis of Alzheimer's disease

The causes of neuronal dysfunction and degeneration in Alzheimers disease (AD) are not fully understood, but increased production of neurotoxic forms of amyloid β‐peptide‐42 (Aβ42) seems of major importance. Large extracellular deposits of aggregated Aβ42 (plaques) is a diagnostic feature of AD, but Aβ42 may be particularly cytotoxic when it accumulates inside neurons. The factors that may promote the intracellular accumulation of Aβ42 in AD are unknown, but recent findings suggest that individuals with elevated homocysteine levels are at increased risk for AD. We show that homocysteic acid (HA), an oxidized metabolite of homocysteine, induces intraneuronal accumulation of a Aβ42 that is associated with cytotoxicity. The neurotoxicity of HA can be attenuated by an inhibitor of γ‐secretase, the enzyme activity that generates Aβ42, suggesting a key role for intracellular Aβ42 accumulation in the neurotoxic action of HA. Concentrations of HA in cerebrospinal fluid (CSF) were similar in AD and control subjects. CSF homocysteine levels were elevated significantly in AD patients, however, and homocysteine exacerbated HA‐induced neurotoxicity, suggesting a role for HA in the pathogenic action of elevated homocysteine levels in AD. These findings suggest that the intracellular accumulation of Aβ42 plays a role in the neurotoxic action of HA, and suggest a potential therapeutic benefit of agents that modify the production and neurotoxic actions of HA and homocysteine.

Pro-apoptotic effect of presenilin 2 (PS2) overexpression is associated with down-regulation of Bcl-2 in cultured neurons

Presenilin 2 (PS2) is a polytopic membrane protein that is mutated in some cases of familial Alzheimers disease (AD). The normal functions of PS2 and its pathogenic role in AD remain unclear. We investigated the biological role of this protein in neurons, using adenovirus‐mediated transduction of the PS2 gene into rat primary cortical neurons. Immunocytochemical analyses demonstrated increased PS2 immunoreactivity in most neurons infected with recombinant adenoviruses expressing PS2. Neurons infected with wild‐type or mutant (N141I) PS2‐expressing adenoviruses showed a significant increase in basal cell death, compared with those infected with control β‐galactosidase‐expressing adenovirus. Moreover, PS2 overexpression markedly increased neuronal susceptibility to staurosporine‐induced apoptosis. Mutant PS2 was more effective in enhancing apoptosis than its wild‐type counterpart. Staurosporine‐induced death was significantly inhibited by a specific caspase 3 inhibitor. Western analyses revealed that Bcl‐2 protein expression was specifically down‐regulated in neurons overexpressing PS2, which temporally corresponded to the accumulation of C‐ and N‐terminal fragments of PS2. Additionally, expression of mutant, but not wild‐type PS2, increased the production of β‐amyloid protein (Aβ) 42. These data collectively suggest that the pro‐apoptotic effect of PS2 is mediated by down‐regulation of Bcl‐2. PS2 mutations may increase the susceptibility of neurons to apoptotic stimuli by perturbing the regulation of cell death.

Oxidative stress up-regulates presenilin 1 in lipid rafts in neuronal cells

Oxidative stress is associated with β‐amyloid peptide (Aβ) accumulation in the brains of Alzheimers disease patients. Aβ is generated upon the sequential proteolytic cleavage of transmembrane amyloid precursor protein (APP) by two membrane‐bound proteases, β‐secretase (BACE1) and the γ‐secretase complex comprising presenilin 1 (PS1), nicastrin, APH‐1 and PEN‐2. Recent evidence suggests that significant amounts of BACE1 and γ‐secretase components localize in the cholesterol‐rich region of membranes known as lipid rafts, where Aβ production occurs preferentially. In this study, we investigated the effects of oxidative stress on the BACE1 and γ‐secretase components in lipid rafts using human neuroblastoma SH‐SY5Y cells exposed to ethacrynic acid (EA), a compound that induces cellular glutathione depletion. Following exposure of cells to EA, heme oxygenase‐1, a marker protein of oxidative stress, was strongly induced. Moreover, treatment with EA resulted in a significant increase in PS1 protein levels, but not those of nicastrin, APH‐1, PEN‐2 or BACE1, in both cell lysates and the lipid raft fraction. This increase in PS1 protein expression was prevented by co‐treatment with an antioxidant, N‐acetylcysteine (NAC). EA additionally induced a significant increase in PS1 mRNA expression, which was inhibited by NAC. Finally, EA treatment was found to promote Aβ secretion from cells expressing Swedish mutant APP. It appears that in our cell culture model, oxidative stress enhances PS1 protein levels in lipid rafts via up‐regulation of PS1 transcription, which may constitute the mechanism underlying the oxidative stress‐associated promotion of Aβ production.

Prostaglandin E2 Stimulates the Production of Amyloid-β Peptides through Internalization of the EP4 Receptor

Amyloid-β (Aβ) peptides, generated by the proteolysis of β-amyloid precursor protein by β- and γ-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E2 (PGE2), a strong inducer of inflammation, stimulates the production of Aβ through EP2 and EP4 receptors, and here we have examined the molecular mechanism. Activation of EP2 and EP4 receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP2, but not EP4, receptor-mediated stimulation of the Aβ production. In contrast, inhibitors of endocytosis suppressed EP4, but not EP2, receptor-mediated stimulation. Activation of γ-secretase was observed with the activation of EP4 receptors but not EP2 receptors. PGE2-dependent internalization of the EP4 receptor was observed, and cells expressing a mutant EP4 receptor lacking the internalization activity did not exhibit PGE2-stimulated production of Aβ. A physical interaction between the EP4 receptor and PS-1, a catalytic subunit of γ-secretases, was revealed by immunoprecipitation assays. PGE2-induced internalization of PS-1 and co-localization of EP4, PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP4 receptor null mice. These results suggest that PGE2-stimulated production of Aβ involves EP4 receptor-mediated endocytosis of PS-1 followed by activation of the γ-secretase, as well as EP2 receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease.