Inhibition of microtubule assembly competent tubulin synthesis leads to accumulation of phosphorylated tau in neuronal cell bodies.

Neurofibrillary tangles, a pathological hallmark of Alzheimer's disease (AD), are somatodendritic filamentous inclusions composed of hyperphosphorylated tau. Microtubule loss is also a common feature of affected neurons in AD. However, whether and how the disruptions of microtubules and the microtubule-associated proteins occur in the pathogenesis of AD remain unclear. Recent evidence indicates that reduced expression of tubulin by knocking down a tubulin chaperon can cause tau neurotoxicity. Thus, the disruption of tubulin homeostasis may result in the acquisition of tau pathogenesis and ultimately cause tauopathy. To investigate whether the disruption of tubulin maintenance induces tau abnormalities in mammalian neurons, we developed a miRNA-mediated knockdown system of tubulin-specific chaperon E (Tbce), which is a factor required for the de novo synthesis of tubulin. Tbce knockdown in mouse primary cultured neurons induced an increase in tubulin in the cell body at 14 days in vitro. Accumulated tubulin was not acetylated or incorporated in microtubules, indicating that they were functionally inert. Concomitantly, tau also accumulated in neuronal cell bodies. The mis-localized tau was phosphorylated at Ser202/Thr205 and Ser396/Ser404. These results indicate that Tbce knockdown in mammalian neurons induces not only a reduction in properly folded tubulins, which are microtubule assembly competent, but also an accumulation of phosphorylated tau in the cell body of mammalian neurons. These findings suggest that disruption of the homeostatic mechanism for maintaining tubulin biosynthesis and/or microtubules can cause tau accumulation in the cell body, which is commonly observed in tauopathies.

Regulatory mechanisms for the axonal localization of tau protein in neurons.

Tau is a microtubule (MT)-associated protein that is thought to be localized to the axon. However, its precise localization in developing neurons and mechanisms for the axonal localization have not been fully addressed. In this study, we found that the axonal localization of tau in cultured rat hippocampal neurons mainly occur during early neuronal development. Interestingly, transient expression of human tau in very immature neurons, but not in mature neurons, mimicked the developmental localization of endogenous tau to the axon. We therefore were able to establish an experimental model, in which exogenously expressed tau can be properly localized to the axon. Using this model, we obtained a surprising finding that the axonal localization of tau did not require stable MT binding. Tau lacking the MT-binding domain (MTBD) exhibited high diffusivity but localized properly to the axon. In contrast, a dephosphorylation-mimetic mutant of the proline-rich region 2 showed reinforced MT binding and mislocalization. Our results suggest that tight binding to MTs prevents tau from entering the axon and results in mislocalization in the soma and dendrites when expressed in mature neurons. This study therefore provides a novel mechanism independent of MTBD for the axonal localization of tau.

The A673T mutation in the amyloid precursor protein reduces the production of ß-amyloid protein from its ß-carboxyl terminal fragment in cells.

INTRODUCTION: The A673T mutation in the amyloid precursor protein (APP) protects against Alzheimer's disease by reducing ß-amyloid protein (Aß) production. This mutation reduced the release of the soluble APP fragment (sAPPß), which is processed by ß-secretase, suggesting a concomitant decrease in the ß-carboxyl fragment of APP (C99), which is a direct substrate of γ-secretase for Aß production. However, it remains controversial whether the level of C99 is significantly reduced in cells expressing APP that carry A673T as the cause of reduced Aß production. Here, we investigated the effect of the A673T mutation in C99 on γ-cleavage in cells. RESULTS: We found that the level of C99 in cells expressing APP A673T was indistinctive of that observed in cells expressing wild-type APP, although the release of sAPPß was significantly reduced in the APP A673T cells. In addition, our reconstituted ß-secretase assay demonstrated no significant difference in ß-cleavage on an APP fragment carrying the A673T mutation compared with the wild-type fragment. Importantly, cells expressing C99 containing the A673T mutation (C99 A2T; in accordance with the Aß numbering) produced roughly half the level of Aß compared with the wild-type C99, suggesting that the C99 A2T is an insufficient substrate of γ-secretase in cells. A cell-free γ-secretase assay revealed that Aß production from the microsomal fraction of cells expressing C99 A2T was diminished. A sucrose gradient centrifugation analysis indicated that the levels of the C99 A2T that was codistributed with γ-secretase components in the raft fractions were reduced significantly. CONCLUSIONS: Our data indicate that the A673T mutation in APP alters the release of sAPPß, but not the C99 level, and that the C99 A2T is an inefficient substrate for γ-secretase in cell-based assay.