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.

Ectopic Expression Induces Abnormal Somatodendritic Distribution of Tau in the Mouse Brain.

Tau is a microtubule (MT)-associated protein that is localized to the axon. In Alzheimer's disease, the distribution of tau undergoes a remarkable alteration, leading to the formation of tau inclusions in the somatodendritic compartment. To investigate how this mislocalization occurs, we recently developed immunohistochemical tools that can separately detect endogenous mouse and exogenous human tau with high sensitivity, which allows us to visualize not only the pathological but also the pre-aggregated tau in mouse brain tissues of both sexes. Using these antibodies, we found that in tau-transgenic mouse brains, exogenous human tau was abundant in dendrites and somata even in the presymptomatic period, whereas the axonal localization of endogenous mouse tau was unaffected. In stark contrast, exogenous tau was properly localized to the axon in human tau knock-in mice. We tracked this difference to the temporal expression patterns of tau. Endogenous mouse tau and exogenous human tau in human tau knock-in mice exhibited high expression levels during the neonatal period and strong suppression into the adulthood. However, human tau in transgenic mice was expressed continuously and at high levels in adult animals. These results indicated the uncontrolled expression of exogenous tau beyond the developmental period as a cause of mislocalization in the transgenic mice. Superresolution microscopic and biochemical analyses also indicated that the interaction between MTs and exogenous tau was impaired only in the tau-transgenic mice, but not in knock-in mice. Thus, the ectopic expression of tau may be critical for its somatodendritic mislocalization, a key step of the tauopathy.SIGNIFICANCE STATEMENT Somatodendritic localization of tau may be an early step leading to the neuronal degeneration in tauopathies. However, the mechanisms of the normal axonal distribution of tau and the mislocalization of pathological tau remain obscure. Our immunohistochemical and biochemical analyses demonstrated that the endogenous mouse tau is transiently expressed in neonatal brains, that exogenous human tau expressed corresponding to such tau expression profile can distribute into the axon, and that the constitutive expression of tau into adulthood (e.g., human tau in transgenic mice) results in abnormal somatodendritic localization. Thus, the expression profile of tau is tightly associated with the localization of tau, and the ectopic expression of tau in matured neurons may be involved in the pathogenesis of tauopathy.

Distribution of endogenous normal tau in the mouse brain.

Tau is a microtubule-associated protein (MAP) that is localized to the axon. In Alzheimer's disease (AD), the distribution of tau undergoes a remarkable alteration, leading to the formation of tau inclusions in the somatodendritic compartment. While the abnormal aggregated tau has been extensively studied in human patient tissues and animal models of AD, how normal tau localizes to the axon, which would be the foundation to understand how the mis-localization occurs, has not been well studied due to the poor detectability of normal unaggregated tau in vivo. Therefore, we developed immunohistochemical techniques that can detect normal mouse and human tau in brain tissues with high sensitivity. Using these techniques, we demonstrate the global distribution of tau in the mouse brain and confirmed that normal tau is exclusively localized to the axonal compartment in vivo. Interestingly, tau antibodies strongly labeled nonmyelinated axons such as hippocampal mossy fibers, while white matters generally exhibited low levels of immunoreactivity. Furthermore, mouse tau is highly expressed not only in neurons but also in oligodendrocytes. With super resolution imaging using the stimulated-depletion microscopy, axonal tau appeared punctate rather than fibrous, indicating that tau decorates microtubules sparsely. Co-labeling with presynaptic and postsynaptic markers revealed that normal tau is not localized to synapses but sparsely distributes in the axon. Taken together, this study reports novel antibodies to investigate the localization and mis-localization of tau in vivo and novel findings of normal tau localization in the mouse brain.

Distinct deposition of amyloid-ß species in brains with Alzheimer's disease pathology visualized with MALDI imaging mass spectrometry.

Amyloid ß (Aß) deposition in the brain is an early and invariable feature of Alzheimer's disease (AD). The Aß peptides are composed of about 40 amino acids and are generated from amyloid precursor proteins (APP), by ß- and γ-secretases. The distribution of individual Aß peptides in the brains of aged people, and those suffering from AD and cerebral amyloid angiopathy (CAA), is not fully characterized. We employed the matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) to illustrate the spatial distribution of a broad range of Aß species in human autopsied brains. With technical advancements such as formic acid pretreatment of frozen autopsied brain samples, we have: i) demonstrated that Aß1-42 and Aß1-43 were selectively deposited in senile plaques while full-length Aß peptides such as Aß1-36, 1-37, 1-38, 1-39, 1-40, and Aß1-41 were deposited in leptomeningeal blood vessels. ii) Visualized distinct depositions of N-terminal truncated Aß40 and Aß42, including pyroglutamate modified at Glu-3 (N3pE), only with IMS for the first time. iii) Demonstrated that one single amino acid alteration at the C-terminus between Aß1-42 and Aß1-41 results in profound changes in their distribution pattern. In vitro, this can be attributed to the difference in the self-aggregation ability amongst Aß1-40, Aß1-41, and Aß1-42. These observations were further confirmed with immunohistochemistry (IHC), using the newly developed anti-Aß1-41 antibody. Here, distinct depositions of truncated and/or modified C- and N-terminal fragments of Aßs in AD and CAA brains with MALDI-IMS were visualized in a spacio-temporal specific manner. Specifically, Aß1-41 was detected both with MALDI-IMS and IHC suggesting that a single amino acid alteration at the C-terminus of Aß results in drastic distribution changes. These results suggest that MALDI-IMS could be used as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD and CAA.

γ-Secretase associated with lipid rafts: multiple interactive pathways in the stepwise processing of ß-carboxyl-terminal fragment.

γ-Secretase generates amyloid ß-protein (Aß), a pathogenic molecule in Alzheimer disease, through the intramembrane cleavage of the ß-carboxyl-terminal fragment (ßCTF) of ß-amyloid precursor protein. We previously showed the framework of the γ-secretase cleavage, i.e. the stepwise successive processing of ßCTF at every three (or four) amino acids. However, the membrane integrity of γ-secretase was not taken into consideration because of the use of the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized reconstituted γ-secretase system. Here, we sought to address how the membrane-integrated γ-secretase cleaves ßCTF by using γ-secretase associated with lipid rafts. Quantitative analyses using liquid chromatography-tandem mass spectrometry of the ßCTF transmembrane domain-derived peptides released along with Aß generation revealed that the raft-associated γ-secretase cleaves ßCTF in a stepwise sequential manner, but novel penta- and hexapeptides as well as tri- and tetrapeptides are released. The cropping of these peptides links the two major tripeptide-cleaving pathways generating Aß40 and Aß42 at several points, implying that there are multiple interactive pathways for the stepwise cleavages of ßCTF. It should be noted that Aß38 and Aß43 are generated through three routes, and γ-secretase modulator 1 enhances all the three routes generating Aß38, which results in decreases in Aß42 and Aß43 and an increase in Aß38. These observations indicate that multiple interactive pathways for stepwise successive processing by γ-secretase define the species and quantity of Aß produced.

Cerebrospinal fluid levels of phosphorylated tau and Aß1-38/Aß1-40/Aß1-42 in Alzheimer's disease with PS1 mutations.

We studied seven cases of Alzheimer's disease (AD). Six of the patients had presenilin 1 (PS1) mutations (PS1AD). Three novel PS1 mutations (T99A, H131R and L219R) and three other missense mutations (M233L, H163R and V272A) were found in the PS1AD group. We measured the levels of phosphorylated tau (ptau-181, ptau-199) and Aß (Aß1-42, Aß1-40 and Aß1-38) in the cerebrospinal fluid (CSF) of PS1AD patients, early-onset sporadic AD (EOSAD), late-onset sporadic AD (LOSAD) and non-demented subjects (ND). The CSF levels of Aß1-42 in the three AD groups were significantly lower than those of the ND group (p < 0.0001). CSF levels of Aß1-42 in the PS1AD group were significantly lower than those in the two sporadic AD groups. The Aß1-40 and Aß1-38 levels in the CSF of the PS1AD group were significantly lower than those of the three other groups (p < 0.0001, respectively). The levels of Aß1-40, Aß1-38 and Aß1-42 in the CSF of the PS1AD group remained lower than those of the ND group for 4 years. Not only CSF Aß1-42, but also Aß1-40 and Aß1-38 decreased in the advanced stages of PS1AD.

Encephalopathy with amyloid angiopathy and numerous amyloid plaques with low levels of CSF Aß1-40/Aß1-42.

A middle-aged male suffering from encephalopathy with cerebral amyloid angiopathy (CAA) with amyloid beta (Aß) presented with initial symptoms of transient consciousness disturbance and left visual field photophobia. Lesions with aberrantly high signal on T2-weighted magnetic resonance imaging (MRI) of the brain appeared in the right temporal lobe posterior to the occipital lobe and spread to other areas. Brain biopsy revealed Aß deposits in vascular walls and numerous diffuse plaques in parenchymal areas. Based on MRI findings, Initial corticosteroid therapy with beta methasone effectively improved the neurological symptoms of consciousness disturbance and motor deficits. After corticosteroid therapy was stopped at 4 weeks, recurrence occurred. Additional corticosteroids did not improve clinical symptoms and the patient progressed to a bed-ridden state with a severe consciousness disturbance. Notably, CSF Aß1-42 and CSF Aß1-40 decreased while the recurrent encephalopathy worsened. After intense deterioration, the patient became stable. CSF Aß1-42 increased but remained at a very low level. This case of CAA encephalopathy with apolipoprotein E ϵ4/ϵ4 homozygosity showed Aß deposits in vascular walls and numerous diffuse plaques in parenchymal areas. The clinical course suggests that reduction of CSF Aß1-42 and Aß1-40 might be related to clinical deterioration in cases of encephalopathy.

Phosphorylation-dependent TDP-43 antibody detects intraneuronal dot-like structures showing morphological characters of granulovacuolar degeneration.

TAR-DNA-binding protein 43 (TDP-43) was considered to be a disease-specific component of ubiquitin-positive and tau-negative inclusions in the brains of patients with frontotemporal lobar degeneration and amyotrophic lateral sclerosis (ALS). However, this protein also accumulates abnormally in neurons in other neurodegenerative diseases, including Alzheimer's disease (AD). Although the role of TDP-43 deposition in these diseases is not clear, abnormal phosphorylation of the protein is suggested to be a critical step in disease pathogenesis. In this study, we generated a new phosphorylation-dependent TDP-43 antibody and examined AD brain sections from temporal lobes, including the hippocampus and temporal neocortex, by immunohistochemistry. The antibody, called A2, specifically recognized phosphorylated TDP-43 in western blotting using ALS and AD specimens, detecting a strong 45kDa band and several shorter fragments at around 25kDa with smears. Immunohistochemistry demonstrated neuronal cytoplasmic inclusions in AD brain sections without staining nuclei that were normal physiological TDP-43 localization sites. These results were consistent with previous reports. However, intraneuronal dot-like structures were also intensely labeled by immunohistochemistry. These structures were observed in all the AD brain sections examined and also occurred in sections from the brains of aged subjects without AD pathologies. The morphological and immunohistochemical characteristics of these granular structures were compatible with those of granulovacuolar degeneration (GVD). The A2 antibody clearly and intensely detected granular structures distributed over the hippocampus, subiculum, parahippocampus and temporal neocortex. Thus, immunohistochemistry using phosphorylation-dependent TDP-43 antibodies would be a new useful tool for identifying GVD.

Variations in the effects on synthesis of amyloid beta protein in modulated autophagic conditions.

OBJECTIVE: Autophagy, the intracellular breakdown system for proteins and some organelles, is considered to be important in neurodegenerative disease. Recent reports suggest that autophagy plays an important role in Alzheimer's disease pathogenesis and autophagic vacuoles (AVs) may be sites of amyloid beta protein (Abeta) generation. We attempted to determine if imposed changes in autophagic activity are linked to Abeta generation and secretion in cultured cells. METHODS: We used Chinese hamster ovary cells, stably expressing wild-type APP 751. We treated the cells with three known autophagy modulating conditions, rapamycin treatment, U18666A treatment and cholesterol depletion. RESULTS: All the three conditions resulted in increased levels of LC3-II by western blotting, together with an increase in the number of LC3-positive granules. However, the effects on Abeta production were inconsistent. The rapamycin treatment increased Abeta production and secretion, but the other two conditions had opposite effects. When the level of phosphorylation of the mammalian target of rapamycin (mTOR) was measured, down-regulation of phosphorylated mTOR levels was observed only in rapamycin-treated cells. The LC3-positive granules in the U18666A-treated and cholesterol-depleted cells were different from those in rapamycin-treated cells in terms of number, size and distribution, suggesting that degradative process from autophagosomes to lysosomes was disturbed. DISCUSSION: The biochemical pathways leading to autophagy and the generation of AVs appear to be different in cells treated by the three methods. These differences may explain why the similar autophagic status determined by LC3 immunoreactivities does not correlate with Abeta generation and secretion.

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.