Disulfide bond formation in microtubule-associated tau protein promotes tau accumulation and toxicity in vivo.

Accumulation of microtubule-associated tau protein is thought to cause neuron loss in a group of neurodegenerative diseases called tauopathies. In diseased brains, tau molecules adopt pathological structures that propagate into insoluble forms with disease-specific patterns. Several types of posttranslational modifications in tau are known to modulate its aggregation propensity in vitro, but their influence on tau accumulation and toxicity at the whole-organism level has not been fully elucidated. Herein, we utilized a series of transgenic Drosophila models to compare systematically the toxicity induced by five tau constructs with mutations or deletions associated with aggregation, including substitutions at seven disease-associated phosphorylation sites (S7A and S7E), deletions of PHF6 and PHF6* sequences (ΔPHF6 and ΔPHF6*), and substitutions of cysteine residues in the microtubule binding repeats (C291/322A). We found that substitutions and deletions resulted in different patterns of neurodegeneration and accumulation, with C291/322A having a dramatic effect on both tau accumulation and neurodegeneration. These cysteines formed disulfide bonds in mouse primary cultured neurons and in the fly retina, and stabilized tau proteins. Additionally, they contributed to tau accumulation under oxidative stress. We also found that each of these cysteine residues contributes to the microtubule polymerization rate and microtubule levels at equilibrium, but none of them affected tau binding to polymerized microtubules. Since tau proteins expressed in the Drosophila retina are mostly present in the early stages of tau filaments self-assembly, our results suggest that disulfide bond formation by these cysteine residues could be attractive therapeutic targets.

Quantitative fractionation of tissue microtubules with distinct biochemical properties reflecting their stability and lability.

Microtubules form a major cytoskeleton and exhibit dynamic instability through the repetitive polymerization/depolymerization of tubulin dimers. Although microtubule stability should be precisely controlled to maintain various cellular functions, it has been difficult to assess its status in vivo. Here, we propose a tubulin fractionation method reflecting the stability of microtubules in mouse tissues. Analyses of tubulin fractionated by two-step of ultracentrifugation demonstrated three distinct pools of tubulin, that appeared to be stable microtubule, labile microtubule, and free tubulin. Using this method, we were able to show the specific binding of different microtubule-associated proteins onto each pool of microtubules. Also, there were clear differences in the population of stable microtubule among tissues depending on the proliferative capacity of the constituent cells. These findings indicate that this method is useful for broad analysis of microtubule stability in physiological and pathological conditions.

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.

Glycosylation status of nicastrin influences catalytic activity and substrate preference of γ-secretase.

γ-Secretase complex, the assembly of nicastrin (NCT), Presenilin (PS), Presenilin Enhancer-2 (PEN-2) and Anterior pharynx defective 1 (Aph-1), catalyzes the cleavage of amyloid precursor protein to generate amyloid-ß protein (Aß), the main culprit of Alzheimer's disease. NCT becomes matured through complex glycosylation and play important role in γ-secretase activity by interacting with catalytic subunit PS. However, the role of NCT glycosylation on γ-secretase activity and substrate specificity is still unknown. The purpose of this study is to investigate the effect of NCT glycosylation on γ-secretase activity and substrate specificity in a group of glycosylation mutant lectin resistant CHO (Lec) cells. CHO Lec-1 cells lack glycosyltransferase-I, GnT-I, thus N-glycan on NCT are all oligomannose type, whereas CHO Lec-2 cells synthesize NCT containing sialic acid deficient oligosaccharides due to the impairment of cytidine 5'-monophosphate-sialic acid transporter. Here, we reported that mutant CHO Lec-1 and Lec-2 reduced γ-secretase activity in both cell-based and biochemical assays, and that CHO Lec-1 preferentially reduced Aß generation. Endogenous level of γ-secretase complex, subcellular distribution of γ-secretase subunits and the level of functional γ-secretase complex remained unchanged in mutants. Interestingly, Coimmunoprecipitation study revealed that mutant γ-secretase could recognize substrate as well as parental γ-secretase. Our data suggests that thorough glycosylation of NCT is critical for enzymatic activity and substrate preference of γ-secretase.

Alanine substitutions in the GXXXG motif alter C99 cleavage by γ-secretase but not its dimerization.

The amyloid ß (Aß) protein is a major component of senile plaques, one of the neuropathological hallmarks of Alzheimer's disease. Amyloidogenic processing of amyloid precursor protein (APP) by ß- and γ-secretases leads to production of Aß. APP contains tandem triple repeats of the GXXXG motif in its extracellular juxtamembrane and transmembrane regions. It is reported that the GXXXG motif is related to protein-protein interactions, but it remains controversial whether the GXXXG motif in APP is involved in substrate dimerization and whether dimerization affects γ-secretase-dependent cleavage. Therefore, the relationship between the GXXXG motifs, substrate dimerization, and γ-secretase-dependent cleavage sites remains unclear. Here, we applied blue native poly acrylamide gel electrophoresis to examine the effect of alanine substitutions within the GXXXG motifs of APP carboxyl terminal fragment (C99) on its dimerization and Aß production. Surprisingly, alanine substitutions in the motif failed to alter C99 dimerization in detergent soluble state. Cell-based and solubilized γ-secretase assays demonstrated that increasing alanine substitutions in the motif tended to decrease long Aß species such as Aß42 and Aß43 and to increase in short Aß species concomitantly. Our data suggest that the GXXXG motif is crucial for Aß production, but not for C99 dimerization.

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.

Substrate ectodomain is critical for substrate preference and inhibition of γ-secretase.

Understanding the substrate recognition mechanism of γ-secretase is a key step for establishing substrate-specific inhibition of amyloid ß-protein (Aß) production. However, it is widely believed that γ-secretase is a promiscuous protease and that its substrate-specific inhibition is elusive. Here we show that γ-secretase distinguishes the ectodomain length of substrates and preferentially captures and cleaves substrates containing a short ectodomain. We also show that a subset of peptides containing the CDCYCxxxxCxCxSC motif binds to the amino terminus of C99 and inhibits Aß production in a substrate-specific manner. Interestingly, these peptides suppress ß-secretase-dependent cleavage of APP, but not that of sialyltransferase 1. Most importantly, intraperitoneal administration of peptides into mice results in a significant reduction in cerebral Aß levels. This report provides direct evidence of the substrate preference of γ-secretase and its mechanism. Our results demonstrate that the ectodomain of C99 is a potent target for substrate-specific anti-Aß therapeutics to combat Alzheimer's disease.

Phosphoinositides suppress gamma-secretase in both the detergent-soluble and -insoluble states.

gamma-Secretase is an aspartic protease that hydrolyzes type I membrane proteins within the hydrophobic environment of the lipid bilayer. Using the CHAPSO-solubilized gamma-secretase assay system, we previously found that gamma-secretase activity was sensitive to the concentrations of detergent and phosphatidylcholine. This strongly suggests that the composition of the lipid bilayer has a significant impact on the activity of gamma-secretase. Recently, level of secreted beta-amyloid protein was reported to be attenuated by increasing levels of phosphatidylinositol 4,5-diphosphate (PI(4,5)P2) in cultured cells. However, it is not clear whether PI(4,5)P2 has a direct effect on gamma-secretase activity. In this study, we found that phosphoinositides directly inhibited CHAPSO-solubilized gamma-secretase activity. Interestingly, neither phosphatidylinositol nor inositol triphosphate altered gamma-secretase activity. PI(4,5)P2 was also found to inhibit gamma-secretase activity in CHAPSO-insoluble membrane microdomains (rafts). Kinetic analysis of beta-amyloid protein production in the presence of PI(4,5)P2 suggested a competitive inhibition. Even though phosphoinositides are minor phospholipids of the membrane, the concentration of PI(4,5)P2 within the intact membrane has been reported to be in the range of 4-8 mm. The presence of PI(4,5)P2-rich rafts in the membrane has been reported in a range of cell types. Furthermore, gamma-secretase is enriched in rafts. Taking these data together, we propose that phosphoinositides potentially regulate gamma-secretase activity by suppressing its association with the substrate.