Akiko Oda

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.

IGF-1 promotes β-amyloid production by a secretase-independent mechanism

Beta-amyloid peptide (Abeta) is generated via the sequential proteolysis of beta-amyloid precursor protein (APP) by beta- and gamma-secretases, and plays a crucial role in the pathogenesis of Alzheimers disease (AD). Here, we sought to clarify the role of insulin-like growth factor-1 (IGF-1), implicated in the AD pathomechanism, in the generation of Abeta. Treatment of neuroblastoma SH-SY5Y cells expressing AD-associated Swedish mutant APP with IGF-1 did not alter cellular levels of APP, but significantly increased those of beta-C-terminal fragment (beta-CTF) and secreted Abeta. IGF-1 also enhanced APP phosphorylation at Thr668. Treatment of beta-CTF-expressing cells with IGF-1 increased the levels of beta-CTF and secreted Abeta. The IGF-1-induced augmentation of beta-CTF was observed in the presence of gamma-secretase inhibitors, but not in cells expressing beta-CTF with a Thr668 to alanine substitution. These results suggest that IGF-1 promotes Abeta production through a secretase-independent mechanism involving APP phosphorylation.

Neuronal β-amyloid generation is independent of lipid raft association of β-secretase BACE1: analysis with a palmitoylation-deficient mutant

β‐Secretase, BACE1 is a neuron‐specific membrane‐associated protease that cleaves amyloid precursor protein (APP) to generate β‐amyloid protein (Aβ). BACE1 is partially localized in lipid rafts. We investigated whether lipid raft localization of BACE1 affects Aβ production in neurons using a palmitoylation‐deficient mutant and further analyzed the relationship between palmitoylation of BACE1 and its shedding and dimerization. We initially confirmed that BACE1 is mainly palmitoylated at four C‐terminal cysteine residues in stably transfected neuroblastoma cells. We found that raft localization of mutant BACE1 lacking the palmitoylation modification was markedly reduced in comparison to wild‐type BACE1 in neuroblastoma cells as well as rat primary cortical neurons expressing BACE1 via recombinant adenoviruses. In primary neurons, expression of wild‐type and mutant BACE1 enhanced production of Aβ from endogenous or overexpressed APP to similar extents with the β‐C‐terminal fragment (β‐CTF) of APP mainly distributed in nonraft fractions. Similarly, β‐CTF was recovered mainly in nonraft fractions of neurons expressing Swedish mutant APP only. These results show that raft association of BACE1 does not influence β‐cleavage of APP and Aβ production in neurons, and support the view that BACE1 cleaves APP mainly in nonraft domains. Thus, we propose a model of neuronal Aβ generation involving mobilization of β‐CTF from nonraft to raft domains. Additionally, we obtained data indicating that palmitoylation plays a role in BACE1 shedding but not dimerization.

Reduction of β-amyloid accumulation by reticulon 3 in transgenic mice.

Inhibition of the β-secretase, BACE1, which cleaves amyloid precursor protein (APP) to produce β-amyloid protein (Aβ), is thought to be a feasible therapeutic strategy for Alzheimers disease. Reticulon (RTN) proteins such as RTN3 have been identified as membrane proteins that interact with BACE1 and inhibit its Aβ-generating activity. In this study, we investigated whether RTN3 can regulate Aβ production in vivo, using transgenic (Tg) mice expressing APP with Swedish and London mutations (APP Tg mice) and those expressing RTN3; the latter mice showed ~1.4-fold higher expression levels of RTN3 protein in the cerebral cortex than non-Tg controls. We analyzed the brains of single APP Tg and double APP/RTN3 Tg mice at the age of approximately 15 months. The levels of secreted APP-β, a direct BACE1 cleavage product of APP, in Tris-soluble fraction were considerably reduced in the hippocampus and cerebral cortex of APP/RTN3 Tg mice relative to those in APP Tg mice. Immunohistochemical analyses demonstrated that Aβ burden and plaques were significantly (by approximately 50%) decreased in both the hippocampus and cerebral cortex of double Tg mice compared to APP Tg mice. Furthermore, the levels of guanidine-soluble Aβ40 and Aβ42 in these brain regions of APP/RTN3 Tg mice were relatively lower than those in APP Tg mice. These findings indicate that even a small increase in RTN3 expression exerts suppressive effects on amyloidogenic processing of APP and Aβ accumulation through modulation of BACE1 activity in vivo, and suggest that induction of RTN3 might be an effective therapeutic strategy against Alzheimers disease.

MEK inhibitors suppress β-amyloid production by altering the level of a β-C-terminal fragment of amyloid precursor protein in neuronal cells

β‐Amyloid peptide (Aβ) is generated via sequential proteolysis of amyloid precursor protein (APP) by β‐ and γ‐secretases. Cell‐based screening experiments disclosed that the MEK (MAP kinase kinase) inhibitors, U0126 and PD184352, suppress Aβ secretion from human neuronal SH‐SY5Y cells expressing Swedish mutant APP. These inhibitors did not affect the cellular levels of APP but significantly reduced those of the APP β‐C‐terminal fragment (β‐CTF). Additionally, β‐CTF levels were markedly reduced by these inhibitors in cells expressing the fragment in a γ‐secretase‐independent and proteasome‐dependent manner. Our results suggest that MEK inhibitors reduce Aβ generation via secretase‐independent alteration of β‐CTF levels.