Clioquinol Decreases Levels of Phosphorylated, Truncated, and Oligomerized Tau Protein.

The neuropathological hallmarks of Alzheimer's disease (AD) are senile plaques (SPs), which are composed of amyloid ß protein (Aß), and neurofibrillary tangles (NFTs), which consist of highly phosphorylated tau protein. As bio-metal imbalance may be involved in the formation of NFT and SPs, metal regulation may be a direction for AD treatment. Clioquinol (CQ) is a metal-protein attenuating compound with mild chelating effects for Zn2+ and Cu2+, and CQ can not only detach metals from SPs, but also decrease amyloid aggregation in the brain. Previous studies suggested that Cu2+ induces the hyperphosphorylation of tau. However, the effects of CQ on tau were not fully explored. To examine the effects of CQ on tau metabolism, we used a human neuroblastoma cell line, M1C cells, which express wild-type tau protein (4R0N) via tetracycline-off (TetOff) induction. In a morphological study and ATP assay, up to 10 µM CQ had no effect on cell viability; however, 100 µM CQ had cytotoxic effects. CQ decreased accumulation of Cu+ in the M1C cells (39.4% of the control), and both total and phosphorylated tau protein. It also decreased the activity of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) (37.3% and 60.7% levels of the control, respectively), which are tau kinases. Of note, activation of protein phosphatase 2A (PP2A), which is a tau phosphatase, was also observed after CQ treatment. Fractionation experiments demonstrated a reduction of oligomeric tau in the tris insoluble, sarkosyl soluble fraction by CQ treatment. CQ also decreased caspase-cleaved tau, which accelerated the aggregation of tau protein. CQ activated autophagy and proteasome pathways, which are considered important for the degradation of tau protein. Although further studies are needed to elucidate the mechanisms responsible for the effects of CQ on tau, CQ may shed light on possible AD therapeutics.

Insulin deficiency promotes formation of toxic amyloid-ß42 conformer co-aggregating with hyper-phosphorylated tau oligomer in an Alzheimer's disease model.

The toxic conformer of amyloid ß-protein (Aß) ending at 42 (Aß42), which contains a unique turn conformation at amino acid residue positions 22 and 23 and tends to form oligomers that are neurotoxic, was reported to play a critical role in the pathomechanisms of Alzheimer's disease (AD), in which diabetes mellitus (DM)-like mechanisms are also suggested to be operative. It remains to be established whether the attenuation of insulin signaling is involved in an increase of toxic Aß42 conformer levels. The present study investigated the association between impaired insulin metabolism and formation of toxic Aß42 conformers in the brains of an AD mouse model. In particular, we studied whether insulin deficiency or resistance affected the formation of toxic Aß42 conformers in vivo. We induced insulin deficiency and resistance in 3xTg-AD mice, a mouse AD model harboring two familial AD-mutant APP (KM670/671NL) and PS1 (M146 V) genes and a mutant TAU (P301L) gene, by streptozotocin (STZ) injection and a high fructose diet (HFuD), respectively. Cognitive impairment was significantly worsened by STZ injection but not by HFuD. Dot blot analysis revealed significant increases in total Aß42 levels and the ratio of toxic Aß42 conformer/total Aß42 in STZ-treated mice compared with control and HFuD-fed mice. Immunostaining showed the accumulation of toxic Aß42 conformers and hyper-phosphorylated tau protein (p-tau), which was more prominent in the cortical and hippocampal neurons of STZ-treated mice compared with HFuD-fed and control mice. HFuD-fed mice showed only a mild-to-moderate increase of these proteins compared with controls. Toxic Aß42 conformers were co-localized with p-tau oligomers (Pearson's correlation coefficient = 0.62) in the hippocampus, indicating their co-aggregation. Toxic Aß42 conformer levels were inversely correlated with pancreatic insulin secretion capacity as shown by fasting immunoreactive insulin levels in STZ-treated mice (correlation coefficient = -0.5879, p = .04441), but not HFuD-fed mice, suggesting a decrease in serum insulin levels correlates with toxic Aß42 conformer formation. Levels of p-Akt and phosphorylated glycogen synthase kinase-3ß measured by a homogeneous time-resolved fluorescence assay were significantly lower in STZ-treated mice than in HFuD-fed mice, suggesting a greater inhibition of brain insulin signaling by STZ than HFuD, although both levels were significantly decreased in these groups compared with controls. Iba1-positive and NOS2-positive areas in the cortex and hippocampus were significantly increased in STZ-treated mice and to a lesser extent in HFuD-fed mice compared with controls. These findings suggest that insulin deficiency rather than insulin resistance and the resultant impairment of brain insulin signaling facilitates the formation of toxic Aß42 conformer and its co-aggregation with p-tau oligomers, and that insulin deficiency is an important pathogenic factor in the progression of AD.

Internalized Tau Oligomers Cause Neurodegeneration by Inducing Accumulation of Pathogenic Tau in Human Neurons Derived from Induced Pluripotent Stem Cells.

Neuronal inclusions of hyperphosphorylated and aggregated tau protein are a pathological hallmark of several neurodegenerative tauopathies, including Alzheimer's disease (AD). The hypothesis of tau transmission in AD has emerged from histopathological studies of the spatial and temporal progression of tau pathology in postmortem patient brains. Increasing evidence in cellular and animal models supports the phenomenon of intercellular spreading of tau. However, the molecular and cellular mechanisms of pathogenic tau transmission remain unknown. The studies described herein investigate tau pathology propagation using human neurons derived from induced pluripotent stem cells. Neurons were seeded with full-length human tau monomers and oligomers and chronic effects on neuronal viability and function were examined over time. Tau oligomer-treated neurons exhibited an increase in aggregated and phosphorylated pathological tau. These effects were associated with neurite retraction, loss of synapses, aberrant calcium homeostasis, and imbalanced neurotransmitter release. In contrast, tau monomer treatment did not produce any measureable changes. This work supports the hypothesis that tau oligomers are toxic species that can drive the spread of tau pathology and neurodegeneration. SIGNIFICANCE STATEMENT: Several independent studies have implicated tau protein as central to Alzheimer's disease progression and cell-to-cell pathology propagation. In this study, we investigated the ability of different tau species to propagate pathology in human neurons derived from induced pluripotent stem cells, which to date has not been shown. We demonstrated that tau oligomers, but not monomers, induce accumulation of pathological, hyperphosphorylated tau. This effect was accompanied with neurite degeneration, loss of synapses, aberrant calcium homeostasis, imbalanced neurotransmitter release, and ultimately with neuronal death. This study bridges various tau pathological phenotypes into a single and relevant induced pluripotent stem cell neuronal model of human disease that can be applied to the discovery of the mechanisms of tau-induced neurodegeneration.

Pioglitazone prevents tau oligomerization.

Tau aggregation and amyloid ß protein (Aß) deposition are the main causes of Alzheimer's disease (AD). Peroxisome proliferator-activated receptor γ (PPARγ) activation modulates Aß production. To test whether the PPARγ agonist pioglitazone (PIO) is also effective in preventing tau aggregation in AD, we used a cellular model in which wild-type tau protein (4R0N) is overexpressed (M1C cells) (Hamano et al., 2012) as well as primary neuronal cultures. PIO reduced both phosphorylated and total tau levels, and inactivated glycogen synthase kinase 3ß, a major tau kinase, associated with activation of Akt. In addition, PIO decreased cleaved caspase3 and C-terminal truncated tau species by caspase, which is expected to decrease tau aggregation. A fractionation study showed that PIO reduced high molecular-weight (120 kDa), oligomeric tau species in Tris Insoluble, sarkosyl-soluble fractions. Tau decrease was reversed by adding GW9662, a PPARγ antagonist. Together, our current results support the idea that PPARγ agonists may be useful therapeutic agents for AD.

Nasal Rifampicin Halts the Progression of Tauopathy by Inhibiting Tau Oligomer Propagation in Alzheimer Brain Extract-Injected Mice.

The cell-to-cell transmission of tau aggregates is considered a mechanism underlying the intracerebral spreading of tau pathology in Alzheimer's disease (AD) and other tauopathies. Recent studies suggest that tau oligomers, rather than fibrils, participate in this process. We previously showed that intranasal rifampicin inhibits tau oligomer accumulation and improves cognition in tauopathy mice. In the present study, we examined the effects of nasal rifampicin on tau propagation in a new mouse model of tauopathy. A tau oligomer-rich fraction prepared from the brain of an AD patient was injected into a unilateral hippocampus of tau264 mice that express both 3-repeat and 4-repeat wild-type human tau. Rifampicin administration was started one week after the injection and performed three times a week for 24 weeks. Cognitive function and tau pathology were assessed by the Morris water maze test and brain section staining. Rifampicin treatment inhibited the spreading of tau oligomers from the injection site to other brain regions and neurofibrillary tangle formation in the entorhinal cortex. Synapse and neuronal loss in the hippocampus were also prevented, and cognitive function remained normal. These results suggest that intranasal rifampicin could be a promising remedy that halts the progression of tauopathy by inhibiting tau oligomer propagation.

Tau oligomers accumulation sensitizes prostate cancer cells to docetaxel treatment.

PURPOSE: Human tau is a highly dynamic, multifunctional protein expressed in different isoforms and conformers, known to modulate microtubule turnover. Tau oligomers are considered pathologic forms of the protein able to initiate specific protein accumulation diseases, called tauopathies. In our study, we investigated the potential association between autophagy and tau oligomers accumulation and its role in the response of prostate cancer cells to docetaxel. METHODS: We evaluated in vitro the expression of tau oligomers in prostate cancer cell lines, PC3 and DU145, in presence of autophagy inhibitors and investigated the role of tau oligomers accumulation in resistance to docetaxel treatment. RESULTS: Tau protein was basally expressed in prostate cancer lines as several monomeric and oligomeric forms. The pharmacologic inhibition of autophagy induced in cancer cells the accumulation of tau protein, with a prevalent expression of oligomeric forms. Immunofluorescence analysis of untreated cells revealed that tau was visible mainly in dividing cells where it was localized on the mitotic spindle. Inhibition of autophagy determined an evident upregulation of tau signal in dividing cells and the presence of aberrant monoastral mitotic spindles. The accumulation of tau oligomers was associated with DNA DSB and increased cytotoxic effect by docetaxel. CONCLUSIONS: Our data indicate that autophagy could exert a promoting role in cancer growth and during chemotherapy facilitating degradation of tau protein and thus blocking the antimitotic effect of accumulated tau oligomers. Thus, therapeutic strategies aimed at stimulating tau oligomers formation, such as autophagy inhibition, could be an effective adjuvant in cancer therapy.

Early alterations of neurovascular unit in the retina in mouse models of tauopathy.

The retina, as the only visually accessible tissue in the central nervous system, has attracted significant attention for evaluating it as a biomarker for neurodegenerative diseases. Yet, most of studies focus on characterizing the loss of retinal ganglion cells (RGCs) and degeneration of their axons. There is no integrated analysis addressing temporal alterations of different retinal cells in the neurovascular unit (NVU) in particular retinal vessels. Here we assessed NVU changes in two mouse models of tauopathy, P301S and P301L transgenic mice overexpressing the human tau mutated gene, and evaluated the therapeutic effects of a tau oligomer monoclonal antibody (TOMA). We found that retinal edema and breakdown of blood-retina barrier were observed at the very early stage of tauopathy. Leukocyte adhesion/infiltration, and microglial recruitment/activation were constantly increased in the retinal ganglion cell layer of tau transgenic mice at different ages, while Müller cell gliosis was only detected in relatively older tau mice. Concomitantly, the number and function of RGCs progressively decreased during aging although they were not considerably altered in the very early stage of tauopathy. Moreover, intrinsically photosensitive RGCs appeared more sensitive to tauopathy. Remarkably, TOMA treatment in young tau transgenic mice significantly attenuated vascular leakage, inflammation and RGC loss. Our data provide compelling evidence that abnormal tau accumulation can lead to pathology in the retinal NVU, and vascular alterations occur more manifest and earlier than neurodegeneration in the retina. Oligomeric tau-targeted immunotherapy has the potential to treat tau-induced retinopathies. These data suggest that retinal NVU may serve as a potential biomarker for diagnosis and staging of tauopathy as well as a platform to study the molecular mechanisms of neurodegeneration.

Rho-kinase ROCK inhibitors reduce oligomeric tau protein.

Neurofibrillary tangles, one of the pathological hallmarks of Alzheimer's disease, consist of highly phosphorylated tau proteins. Tau protein binds to microtubules and is best known for its role in regulating microtubule dynamics. However, if tau protein is phosphorylated by activated major tau kinases, including glycogen synthase kinase 3ß or cyclin-dependent kinase 5, or inactivated tau phosphatase, including protein phosphatase 2A, its affinity for microtubules is reduced, and the free tau is believed to aggregate, thereby forming neurofibrillary tangles. We previously reported that pitavastatin decreases the total and phosphorylated tau protein using a cellular model of tauopathy. The reduction of tau was considered to be due to Rho-associated coiled-coil protein kinase (ROCK) inhibition by pitavastatin. ROCK plays important roles to organize the actin cytoskeleton, an expected therapeutic target of human disorders. Several ROCK inhibitors are clinically applied to prevent vasospasm postsubarachnoid hemorrhage (fasudil) and for the treatment of glaucoma (ripasudil). We have examined the effects of ROCK inhibitors (H1152, Y-27632, and fasudil [HA-1077]) on tau protein phosphorylation in detail. A human neuroblastoma cell line (M1C cells) that expresses wild-type tau protein (4R0N) by tetracycline-off (TetOff) induction, primary cultured mouse neurons, and a mouse model of tauopathy (rTG4510 line) were used. The levels of phosphorylated tau and caspase-cleaved tau were reduced by the ROCK inhibitors. Oligomeric tau levels were also reduced by ROCK inhibitors. After ROCK inhibitor treatment, glycogen synthase kinase 3ß, cyclin-dependent kinase 5, and caspase were inactivated, protein phosphatase 2A was activated, and the levels of IFN-γ were reduced. ROCK inhibitors activated autophagy and proteasome pathways, which are considered important for the degradation of tau protein. Collectively, these results suggest that ROCK inhibitors represent a viable therapeutic route to reduce the pathogenic forms of tau protein in tauopathies, including Alzheimer's disease.

Visualization of soluble tau oligomers in TauP301L-BiFC transgenic mice demonstrates the progression of tauopathy.

Accumulation of abnormal tau aggregates in the brain is a pathological hallmark of multiple neurodegenerative disorders including Alzheimer's disease. Increasing evidence suggests that soluble tau aggregates play a key role in tau pathology as neurotoxic species causing neuronal cell death and act as prion-like seeds mediating tau propagation. Despite the pathological relevance, there is a paucity of methods to monitor tau oligomerization in the brain. As a tool to monitor tau self-assembly in the brain, we generated a novel tau transgenic mouse, named TauP301L-BiFC. By introducing bimolecular fluorescence complementation technique to human tau containing a P301L mutation, we were able to monitor and quantify tau self-assembly, represented by BiFC fluorescence in the brains of transgenic TauP301L-BiFC mice. TauP301L-BiFC mice showed soluble tau oligomerization from 3 months, showing significantly enriched BiFC fluorescence in the brain. Then, massive tau fragmentation occured at 6 months showing dramatically decreased TauP301L-BiFC fluorescence. The fragmented tau species served as a seed for insoluble tau aggregation. In a result, insoluble TauP301L-BiFC aggregates coaggregated with endogenous mouse tau accumulated in the brain, showing subsequently increased BiFC fluorescence from 9 months. Neuronal degeneration and cognitive deficits were observed from 12 months of age. TauP301L-BiFC mouse model demonstrated that methylene blue reduced the amount of soluble tau oligomers in the brain, resulting in the prevention of cognitive impairments. We assure that TauP301L-BiFC mice are a bona-fide animal tool to monitor pathological tau oligomerization in AD and other tauopathies.

Tau Oligomers and Fibrils Exhibit Differential Patterns of Seeding and Association With RNA Binding Proteins.

Tau aggregates are pleiotropic and exhibit differences in conformation, structure, and size. These aggregates develop endogenously but are also propagated among neurons in disease. We explored the actions of two distinct types of tau aggregates, tau oligomers (oTau) and tau fibrils (fTau), using a seeding assay in primary neuron cultures expressing human 4R0N tau. We find that oTau and fTau elicit distinct patterns of tau inclusions in the neurons and distinct molecular interactions. The exogenously applied oTau and fTau both clear rapidly from the neurons, but both also seed intracellular inclusions composed of endogenously produced tau. The two types of seeds elicit differential dose-response relationships for seed uptake and the number of resulting intracellular inclusions. Immunocytochemical studies show that co-localization with RNA binding proteins associated with stress granules is much greater for seeds composed of oTau than fTau. Conversely, co-localization with p62/SQSTM1 and thioflavine S is much greater for fTau than oTau. These results suggest that oTau seeds inclusions that modulate the translational stress response and are physiologically active, whereas fTau seeds inclusions that are fibrillar and shunted to the autolysosomal cascade.

Cornel iridoid glycoside induces autophagy to protect against tau oligomer neurotoxicity induced by the activation of glycogen synthase kinase-3ß.

Tau oligomers are the etiologic molecules of Alzheimer's disease, and correlate strongly with neuronal loss and exhibit neurotoxicity. Recent evidence indicates that small tau oligomers are the most relevant toxic aggregate species. The aim of the present study was to investigate the mechanisms of cornel iridoid glycoside (CIG) on tau oligomers and cognitive functions. We injected wortmannin and GF-109203X (WM/GFX, 200 µM each) into the lateral ventricles to induce tau oligomer and memory impairment in rats. When orally administered with CIG at 60 and 120 mg/kg/day for 14 days, CIG decreased the escape latency in the Morris water maze test. We also found that CIG restored the expression of presynaptic p-synapsin, synaptophysin, and postsynaptic density-95 (PSD-95) decreased by WM/GFX in rat cortex. CIG reduced the accumulation of tau oligomers in the brain of WM/GFX rats and in cells transfected with wild type glycogen synthase kinase-3ß (wtGSK-3ß). In addition, CIG up-regulated the levels of ATG7, ATG12, Beclin-1, and LC3II in vivo and in vitro, suggesting the restoration of autophagy function. These results suggest that CIG could ameliorate memory deficits and regulate memory-associated synaptic proteins through the clearance of tau oligomers accumulation. Moreover, CIG clears tau oligomers by restoring autophagy function.

Methylene Blue Inhibits Formation of Tau Fibrils but not of Granular Tau Oligomers: A Plausible Key to Understanding Failure of a Clinical Trial for Alzheimer's Disease.

Alzheimer's disease pathology is characterized by extracellular deposits of amyloid-ß (Aß) and intracellular inclusions of hyperphosphorylated tau. Although genetic studies of familial Alzheimer's disease suggest a causal link between Aß and disease symptoms, the failure of various Aß-targeted strategies to slow or halt disease progression has led to consideration of the idea that inhibition of tau aggregation might be a more promising therapeutic approach. Methylene blue (MB), which inhibits tau aggregation and rescue memory deficits in a mouse model of tauopathy, however, lacked efficacy in a recent Phase III clinical trial. In order to gain insight into this failure, the present study was designed to examine the mechanism through which MB inhibits tau aggregation. We found that MB inhibits heparin-induced tau aggregation in vitro, as measured by thioflavin T fluorescence. Further, MB reduced the amount of tau in precipitants recovered after ultracentrifugation of the aggregation mixture. Atomic force microscopy revealed that MB reduces the number of tau fibrils but increases the number of granular tau oligomers. The latter result was confirmed by sucrose gradient centrifugation: MB treatment was associated with higher levels of granular tau oligomers (fraction 3) and lower levels of tau fibrils (fractions 5 and 6). We previously demonstrated that the formation of granular tau oligomers, rather than tau fibrils, is essential for neuronal death. Thus, the fact that MB actions are limited to inhibition of tau fibril formation provides a mechanistic explanation for the poor performance of MB in the recent Phase III clinical trial.

Heme Oxygenase 1 Induces Tau Oligomer Formation and Synapse Aberrations in Hippocampal Neurons.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by behavioral changes and cognitive decline. Recent evidence suggests that it is the soluble forms of tau oligomers (Tau-O) and Aß oligomers (oAß) rather than the well-studied insoluble protein aggregates that possess the neurotoxicity, infectivity, and amplification underlying disease progression. Heme oxygenase 1 (HO-1), an inducible enzyme upregulated in the cortex and hippocampus of AD brains, was reported to damage neural structures and disrupt brain function, suggesting possible contributions to Tau-O-mediated neurodegeneration. In this study, we focused on the effects of HO-1 on Tau-O formation. In hippocampus of HO-1-overexpressing transgenic mice and neural 2a (N2a) cells, Tau-O was co-localized with HO-1 as visualized by immunofluorescence staining. Furthermore, primary cultured hippocampal neurons from HO-1 transgenic mice showed elevated Tau-O and concomitant reductions in spine density and length as well as dendritic length, diameter, and arborization. Blocking Tau-O formation by isoprenaline reversed these HO-1-induced morphological changes. These results indicated that HO-1 contributes to Tau-O formation and ensuing synaptic damage. Thus, HO-1 is a promising target for AD drug development.

Preparation and Characterization of Tau Oligomer Strains.

An increasing number of studies have demonstrated the existence of multiple conformational entities of tau, as have been observed for prion protein. We have developed and optimized techniques to isolate and study oligomeric tau strains both in vitro and ex vivo. Moreover, we have modified protocols that demonstrate the seeding properties of oligomeric tau strains that are capable of propagating in vivo. These methods and protocols are explained in this chapter.

Tau Oligomers as Pathogenic Seeds: Preparation and Propagation In Vitro and In Vivo.

Tau oligomers have been shown to be the main toxic tau species in a number of neurodegenerative disorders. In order to study tau oligomers both in vitro and in vivo, we have established methods for the reliable preparation, isolation, and detection of tau oligomers. Methods for the seeding of tau oligomers, isolation of tau oligomers from tissue, and detection of tau oligomers using tau oligomer-specific antibodies by biochemical and immunohistochemical methods are detailed below.