Seeding of normal Tau by pathological Tau conformers drives pathogenesis of Alzheimer-like tangles

Neurofibrillary tangles (NFTs) in Alzheimer disease and related tauopathies are composed of insoluble hyperphosphorylated Tau protein, but the mechanisms underlying the conversion of highly soluble Tau into insoluble NFTs remain elusive. Here, we demonstrate that introduction of minute quantities of misfolded preformed Tau fibrils (Tau pffs) into Tau-expressing cells rapidly recruit large amounts of soluble Tau into filamentous inclusions resembling NFTs with unprecedented efficiency, suggesting a "seeding"-recruitment process as a highly plausible mechanism underlying NFT formation in vivo. Consistent with the emerging concept of prion-like transmissibility of disease-causing amyloidogenic proteins, we found that spontaneous uptake of Tau pffs into cells is likely mediated by endocytosis, suggesting a potential mechanism for the propagation of Tau lesions in tauopathy brains. Furthermore, sequestration of soluble Tau by pff-induced Tau aggregates attenuates microtubule overstabilization in Tau-expressing cells, supporting the hypothesis of a Tau loss-of-function toxicity in cells harboring NFTs. In summary, our study establishes a cellular system that robustly develops authentic NFT-like Tau aggregates, which provides mechanistic insights into NFT pathogenesis and a potential tool for identifying Tau-based therapeutics.     

Trans-cellular propagation of Tau aggregation by fibrillar species

Aggregation of the microtubule associated protein Tau is associated with several neurodegenerative disorders, including Alzheimer disease and frontotemporal dementia. In Alzheimer disease, Tau pathology spreads progressively throughout the brain, possibly along existing neural networks. However, it is still unclear how the propagation of Tau misfolding occurs. Intriguingly, in animal models, vaccine-based therapies have reduced Tau and synuclein pathology by uncertain mechanisms, given that these proteins are intracellular. We have previously speculated that trans-cellular propagation of misfolding could be mediated by a process similar to prion pathogenesis, in which fibrillar Tau aggregates spread pathology from cell to cell. However, there has been little evidence to demonstrate true trans-cellular propagation of Tau misfolding, in which Tau aggregates from one cell directly contact Tau protein in the recipient cell to trigger further aggregation. Here we have observed that intracellular Tau fibrils are directly released into the medium and then taken up by co-cultured cells. Internalized Tau aggregates induce fibrillization of intracellular Tau in these naive recipient cells via direct protein-protein contact that we demonstrate using FRET. Tau aggregation can be amplified across several generations of cells. An anti-Tau monoclonal antibody blocks Tau aggregate propagation by trapping fibrils in the extracellular space and preventing their uptake. Thus, propagation of Tau protein misfolding among cells can be mediated by release and subsequent uptake of fibrils that directly contact native protein in recipient cells. These results support the model of aggregate propagation by templated conformational change and suggest a mechanism for vaccine-based therapies in neurodegenerative diseases.     

Mammalian Target of Rapamycin (mTor) Mediates Tau Protein Dyshomeostasis: IMPLICATION FOR ALZHEIMER DISEASE*

Previous evidence from post-mortem Alzheimer disease (AD) brains and drug (especially rapamycin)-oriented in vitro and in vivo models implicated an aberrant accumulation of the mammalian target of rapamycin (mTor) in tangle-bearing neurons in AD brains and its role in the formation of abnormally hyperphosphorylated tau. Compelling evidence indicated that the sequential molecular events such as the synthesis and phosphorylation of tau can be regulated through p70 S6 kinase, the well characterized immediate downstream target of mTor. In the present study, we further identified that the active form of mTor per se accumulates in tangle-bearing neurons, particularly those at early stages in AD brains. By using mass spectrometry and Western blotting, we identified three phosphoepitopes of tau directly phosphorylated by mTor. We have developed a variety of stable cell lines with genetic modification of mTor activity using SH-SY5Y neuroblastoma cells as background. In these cellular systems, we not only confirmed the tau phosphorylation sites found in vitro but also found that mTor mediates the synthesis and aggregation of tau, resulting in compromised microtubule stability. Changes of mTor activity cause fluctuation of the level of a battery of tau kinases such as protein kinase A, v-Akt murine thymoma viral oncogene homolog-1, glycogen synthase kinase 3β, cyclin-dependent kinase 5, and tau protein phosphatase 2A. These results implicate mTor in promoting an imbalance of tau homeostasis, a condition required for neurons to maintain physiological function.     

Extracellular Monomeric Tau Protein Is Sufficient to Initiate the Spread of Tau Protein Pathology

Understanding the formation and propagation of aggregates of the Alzheimer disease-associated Tau protein in vivo is vital for the development of therapeutics for this devastating disorder. Using our recently developed live-cell aggregation sensor in neuron-like cells, we demonstrate that different variants of exogenous monomeric Tau, namely full-length Tau (hTau40) and the Tau-derived construct K18 comprising the repeat domain, initially accumulate in endosomal compartments, where they form fibrillar seeds that subsequently induce the aggregation of endogenous Tau. Using superresolution imaging, we confirm that fibrils consisting of endogenous and exogenous Tau are released from cells and demonstrate their potential to spread Tau pathology. Our data indicate a greater pathological risk and potential toxicity than hitherto suspected for extracellular soluble Tau.     

Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease

Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 μm. The K_i value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 μm. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 μm) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 μm) required to reverse behavioral deficits and pathology in Tau transgenic mice.     

Conformation Determines the Seeding Potencies of Native and Recombinant Tau Aggregates

Intracellular Tau inclusions are a pathological hallmark of several neurodegenerative diseases, collectively known as the tauopathies. They include Alzheimer disease, tangle-only dementia, Pick disease, argyrophilic grain disease, chronic traumatic encephalopathy, progressive supranuclear palsy, and corticobasal degeneration. Tau pathology appears to spread through intercellular propagation, requiring the formation of assembled “prion-like” species. Several cell and animal models have been described that recapitulate aspects of this phenomenon. However, the molecular characteristics of seed-competent Tau remain unclear. Here, we have used a cell model to understand the relationships between Tau structure/phosphorylation and seeding by aggregated Tau species from the brains of mice transgenic for human mutant P301S Tau and full-length aggregated recombinant P301S Tau. Deletion of motifs ²⁷⁵VQIINK²⁸⁰ and ³⁰⁶VQIVYK³¹¹ abolished the seeding activity of recombinant full-length Tau, suggesting that its aggregation was necessary for seeding. We describe conformational differences between native and synthetic Tau aggregates that may account for the higher seeding activity of native assembled Tau. When added to aggregated Tau seeds from the brains of mice transgenic for P301S Tau, soluble recombinant Tau aggregated and acquired the molecular properties of aggregated Tau from transgenic mouse brain. We show that seeding is conferred by aggregated Tau that enters cells through macropinocytosis and seeds the assembly of endogenous Tau into filaments.     

Alternative Conformations of the Tau Repeat Domain in Complex with an Engineered Binding Protein

The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337–342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau.     

Rapid Accumulation of Endogenous Tau Oligomers in a Rat Model of Traumatic Brain Injury: POSSIBLE LINK BETWEEN TRAUMATIC BRAIN INJURY AND SPORADIC TAUOPATHIES*

Traumatic brain injury (TBI) is a serious problem that affects millions of people in the United States alone. Multiple concussions or even a single moderate to severe TBI can also predispose individuals to develop a pathologically distinct form of tauopathy-related dementia at an early age. No effective treatments are currently available for TBI or TBI-related dementia; moreover, only recently has insight been gained regarding the mechanisms behind their connection. Here, we used antibodies to detect oligomeric and phosphorylated Tau proteins in a non-transgenic rodent model of parasagittal fluid percussion injury. Oligomeric and phosphorylated Tau proteins were detected 4 and 24 h and 2 weeks post-TBI in injured, but not sham control rats. These findings suggest that diagnostic tools and therapeutics that target only toxic forms of Tau may provide earlier detection and safe, more effective treatments for tauopathies associated with repetitive neurotrauma.     

Seeded Aggregation and Toxicity of α-Synuclein and Tau: CELLULAR MODELS OF NEURODEGENERATIVE DISEASES*

The deposition of amyloid-like filaments in the brain is the central event in the pathogenesis of neurodegenerative diseases. Here we report cellular models of intracytoplasmic inclusions of α-synuclein, generated by introducing nucleation seeds into SH-SY5Y cells with a transfection reagent. Upon introduction of preformed seeds into cells overexpressing α-synuclein, abundant, highly filamentous α-synuclein-positive inclusions, which are extensively phosphorylated and ubiquitinated and partially thioflavin-positive, were formed within the cells. SH-SY5Y cells that formed such inclusions underwent cell death, which was blocked by small molecular compounds that inhibit β-sheet formation. Similar seed-dependent aggregation was observed in cells expressing four-repeat Tau by introducing four-repeat Tau fibrils but not three-repeat Tau fibrils or α-synuclein fibrils. No aggregate formation was observed in cells overexpressing three-repeat Tau upon treatment with four-repeat Tau fibrils. Our cellular models thus provide evidence of nucleation-dependent and protein-specific polymerization of intracellular amyloid-like proteins in cultured cells.     

Aggregation of detergent-insoluble tau is involved in neuronal loss but not in synaptic loss

Neurofibrillary tangles (NFTs), which consist of highly phosphorylated tau, are hallmarks of neurodegenerative diseases including Alzheimer disease (AD). In neurodegenerative diseases, neuronal dysfunction due to neuronal loss and synaptic loss accompanies NFT formation, suggesting that a process associated with NFT formation may be involved in neuronal dysfunction. To clarify the relationship between the tau aggregation process and synapse and neuronal loss, we compared two lines of mice expressing human tau with or without an aggregation-prone P301L mutation. P301L tau transgenic (Tg) mice exhibited neuronal loss and produced sarcosyl-insoluble tau in old age but did not exhibit synaptic loss and memory impairment. By contrast, wild-type tau Tg mice neither exhibited neuronal loss nor produced sarcosyl-insoluble tau but did exhibit synaptic loss and memory impairment. Moreover, P301L tau was less phosphorylated than wild-type tau, suggesting that the tau phosphorylation state is involved in synaptic loss, whereas the tau aggregation state is involved in neuronal loss. Finally, increasing concentrations of insoluble tau aggregates leads to the formation of fibrillar tau, which causes NFTs to form.     

A Caspase Cleaved Form of Tau Is Preferentially Degraded through the Autophagy Pathway

The microtubule-associated protein tau plays a central role in the pathogenesis of Alzheimer disease (AD) and abnormally accumulates as neurofibrillary tangles; therefore, the pathways by which tau is degraded have been examined extensively. In AD brain tau is abnormally truncated at Asp⁴²¹ (tauΔC), which increases its fibrillogenic properties and results in compromised neuronal function. Given the fact that the accumulation of tauΔC is a pathogenic process in AD, in this study we examined whether full-length tau and tauΔC are degraded through similar or different mechanisms. To this end a tetracycline-inducible model was used to show that tauΔC was degraded significantly faster than full-length tau (FL-tau). Pharmacological inhibition of the proteasome or autophagy pathways demonstrated that although FL-tau is degraded by the proteasome, tauΔC is cleared predominantly by macroautophagy. We also found that tauΔC binds C terminus of Hsp70-interacting protein more efficiently than tau. This interaction leads to an increased ubiquitylation of tauΔC in a reconstituted in vitro assay, but surprisingly, tau (full-length or truncated) was not ubiquitylated in situ. The finding that tauΔC and FL-tau are differentially processed by these degradation systems provides important insights for the development of therapeutic strategies, which are focused on modulating degradation systems to preferentially clear pathological forms of the proteins.     

Interaction of Endogenous Tau Protein with Synaptic Proteins Is Regulated by N-Methyl-D-aspartate Receptor-dependent Tau Phosphorylation

Amyloid-β and tau protein are the two most prominent factors in the pathology of Alzheimer disease. Recent studies indicate that phosphorylated tau might affect synaptic function. We now show that endogenous tau is found at postsynaptic sites where it interacts with the PSD95-NMDA receptor complex. NMDA receptor activation leads to a selective phosphorylation of specific sites in tau, regulating the interaction of tau with Fyn and the PSD95-NMDA receptor complex. Based on our results, we propose that the physiologically occurring phosphorylation of tau could serve as a regulatory mechanism to prevent NMDA receptor overexcitation.     

Characterization of LipL as a non-heme, Fe(II)-dependent α-ketoglutarate:UMP dioxygenase that generates uridine-5'-aldehyde during A-90289 biosynthesis

Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases are a large and diverse superfamily of mononuclear, non-heme enzymes that perform a variety of oxidative transformations typically coupling oxidative decarboxylation of α-KG with hydroxylation of a prime substrate. The biosynthetic gene clusters for several nucleoside antibiotics that contain a modified uridine component, including the lipopeptidyl nucleoside A-90289 from Streptomyces sp. SANK 60405, have recently been reported, revealing a shared open reading frame with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD), a well characterized member of this dioxygenase superfamily. We now provide in vitro data to support the functional assignment of LipL, the putative TauD enzyme from the A-90289 gene cluster, as a non-heme, Fe(II)-dependent α-KG:UMP dioxygenase that produces uridine-5'-aldehyde to initiate the biosynthesis of the modified uridine component of A-90289. The activity of LipL is shown to be dependent on Fe(II), α-KG, and O(2), stimulated by ascorbic acid, and inhibited by several divalent metals. In the absence of the prime substrate UMP, LipL is able to catalyze oxidative decarboxylation of α-KG, although at a significantly reduced rate. The steady-state kinetic parameters using optimized conditions were determined to be K(m)(α-KG) = 7.5 μM, K(m)(UMP) = 14 μM, and k(cat) ≈ 80 min(-1). The discovery of this new activity not only sets the stage to explore the mechanism of LipL and related dioxygenases further but also has critical implications for delineating the biosynthetic pathway of several related nucleoside antibiotics.     

Antibody Uptake into Neurons Occurs Primarily via Clathrin-dependent Fcγ Receptor Endocytosis and Is a Prerequisite for Acute Tau Protein Clearance

Tau immunotherapy is effective in transgenic mice, but the mechanisms of Tau clearance are not well known. To this end, Tau antibody uptake was analyzed in brain slice cultures and primary neurons. Internalization was rapid (<1 h), saturable, and substantial compared with control mouse IgG. Furthermore, temperature reduction to 4 °C, an excess of unlabeled mouse IgG, or an excess of Tau antibodies reduced uptake in slices by 63, 41, and 62%, respectively (p = 0.002, 0.04, and 0.005). Uptake strongly correlated with total and insoluble Tau levels (r² = 0.77 and 0.87 and p = 0.002 and 0.0002), suggesting that Tau aggregates influence antibody internalization and/or retention within neurons. Inhibiting phagocytosis did not reduce uptake in slices or neuronal cultures, indicating limited microglial involvement. In contrast, clathrin-specific inhibitors reduced uptake in neurons (≤78%, p < 0.0001) and slices (≤35%, p = 0.03), demonstrating receptor-mediated endocytosis as the primary uptake pathway. Fluid phase endocytosis accounted for the remainder of antibody uptake in primary neurons, based on co-staining with internalized dextran. The receptor-mediated uptake is to a large extent via low affinity FcγII/III receptors and can be blocked in slices (43%, p = 0.04) and neurons (53%, p = 0.008) with an antibody against these receptors. Importantly, antibody internalization appears to be necessary for Tau reduction in primary neurons. Overall, these findings clarify that Tau antibody uptake is primarily receptor-mediated, that these antibodies are mainly found in neurons with Tau aggregates, and that their intracellular interaction leads to clearance of Tau pathology, all of which have major implications for therapeutic development of this approach.     

Amyloid beta-mediated cell death of cultured hippocampal neurons reveals extensive Tau fragmentation without increased full-length tau phosphorylation

A variety of genetic and biochemical evidence suggests that amyloid β (Aβ) oligomers promote downstream errors in Tau action, in turn inducing neuronal dysfunction and cell death in Alzheimer and related dementias. To better understand molecular mechanisms involved in Aβ-mediated neuronal cell death, we have treated primary rat hippocampal cultures with Aβ oligomers and examined the resulting cellular changes occurring before and during the induction of cell death with a focus on altered Tau biochemistry. The most rapid neuronal responses upon Aβ administration are activation of caspase 3/7 and calpain proteases. Aβ also appears to reduce Akt and Erk1/2 kinase activities while increasing GSK3β and Cdk5 activities. Shortly thereafter, substantial Tau degradation begins, generating relatively stable Tau fragments. Only a very small fraction of full-length Tau remains intact after 4 h of Aβ treatment. In conflict with expectations based on suggested increases of GSK3β and Cdk5 activities, Aβ does not cause any major increases in phosphorylation of full-length Tau as assayed by immunoblotting one-dimensional gels with 11 independent site- and phospho-specific anti-Tau antibodies as well as by immunoblotting two-dimensional gels probed with a pan-Tau antibody. There are, however, subtle and transient increases in Tau phosphorylation at 3-4 specific sites before its degradation. Taken together, these data are consistent with the notion that Aβ-mediated neuronal cell death involves the loss of full-length Tau and/or the generation of toxic fragments but does not involve or require hyperphosphorylation of full-length Tau.     

Spectroscopic Studies of GSK3β Phosphorylation of the Neuronal Tau Protein and Its Interaction with the N-terminal Domain of Apolipoprotein E

Alzheimer disease neurons are characterized by extraneuronal plaques formed by aggregated amyloid-β peptide and by intraneuronal tangles composed of fibrillar aggregates of the microtubule-associated Tau protein. Tau is mostly found in a hyperphosphorylated form in these tangles. Glycogen synthase kinase 3β (GSK3β) is a proline-directed kinase generally considered as one of the major players that (hyper)phosphorylates Tau. The kinase phosphorylates mainly (Ser/Thr)-Pro motifs and is believed to require a priming activity by another kinase. Here, we use an in vitro phosphorylation assay and NMR spectroscopy to characterize in a qualitative and quantitative manner the phosphorylation of Tau by GSK3β. We find that three residues can be phosphorylated (Ser-396, Ser-400, and Ser-404) by GSK3β alone, without priming. Ser-404 is essential in this process, as its mutation to Ala prevents all activity of GSK3β. However, priming enhances the catalytic efficacy of the kinase, as initial phosphorylation of Ser-214 by the cAMP-dependent protein kinase (PKA) leads to the rapid modification by GSK3β of four regularly spaced additional sites. Because the regular incorporation of negative charges by GSK3β leads to a potential parallel between phospho-Tau and heparin, we investigated its interaction with the heparin/low density lipoprotein receptor binding domain of human apolipoprotein E. We indeed observed an interaction between the GSK3β-promoted regular phospho-pattern on Tau and the apolipoprotein E fragment but none in the absence of phosphorylation or the presence of an irregular phosphorylation pattern by the prolonged activity of PKA. Apolipoprotein E is therefore able to discriminate and interact with specific phosphorylation patterns of Tau.     

Specific soluble oligomers of amyloid-β peptide undergo replication and form non-fibrillar aggregates in interfacial environments

Aggregates of amyloid-β (Aβ) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of Aβ aggregates, low molecular weight species ranging between ~2- and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such "off-pathway" 12-18-mer species of Aβ42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we report the physiochemical aspects of LFAO-monomer interactions as well as LFAO-LFAO associations in the presence of interfaces. We discovered that LFAOs are a replicating strain of oligomers that recruit Aβ42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. We also found that in the presence of hexane-buffer or chloroform-buffer interfaces LFAOs are able to associate with themselves to form larger but non-fibrillar aggregates. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. Furthermore, the unique replicating property of off-pathway oligomers may hold profound significance for Alzheimer disease pathology.