Liquid–liquid phase separation of Tau by self and complex coacervation

The liquid–liquid phase separation (LLPS) of Tau has been postulated to play a role in modulating the aggregation property of Tau, a process known to be critically associated with the pathology of a broad range of neurodegenerative diseases including Alzheimer''s Disease. Tau can undergo LLPS by homotypic interaction through self‐coacervation (SC) or by heterotypic association through complex‐coacervation (CC) between Tau and binding partners such as RNA. What is unclear is in what way the formation mechanisms for self and complex coacervation of Tau are similar or different, and the addition of a binding partner to Tau alters the properties of LLPS and Tau. A combination of in vitro experimental and computational study reveals that the primary driving force for both Tau CC and SC is electrostatic interactions between Tau‐RNA or Tau‐Tau macromolecules. The liquid condensates formed by the complex coacervation of Tau and RNA have distinctly higher micro‐viscosity and greater thermal stability than that formed by the SC of Tau. Our study shows that subtle changes in solution conditions, including molecular crowding and the presence of binding partners, can lead to the formation of different types of Tau condensates with distinct micro‐viscosity that can coexist as persistent and immiscible entities in solution. We speculate that the formation, rheological properties and stability of Tau droplets can be readily tuned by cellular factors, and that liquid condensation of Tau can alter the conformational equilibrium of Tau.     

Adult neurogenic process in the subventricular zone‐olfactory bulb system is regulated by Tau protein under prolonged stress

ObjectivesThe area of the subventricular zone (SVZ) in the adult brain exhibits the highest number of proliferative cells, which, together with the olfactory bulb (OB), maintains constant brain plasticity through the generation, migration and integration of newly born neurons. Despite Tau and its malfunction is increasingly related to deficits of adult hippocampal neurogenesis and brain plasticity under pathological conditions [e.g. in Alzheimer''s disease (AD)], it remains unknown whether Tau plays a role in the neurogenic process of the SVZ and OB system under conditions of chronic stress, a well‐known sculptor of brain and risk factor for AD.Materials and methodsDifferent types of newly born cells in SVZ and OB were analysed in animals that lack Tau gene (Tau‐KO) and their wild‐type littermates (WT) under control or chronic stress conditions.ResultsWe demonstrate that chronic stress reduced the number of proliferating cells and neuroblasts in the SVZ leading to decreased number of newborn neurons in the OB of adult WT, but not Tau‐KO, mice. Interestingly, while stress‐evoked changes were not detected in OB granular cell layer, Tau‐KO exhibited increased number of mature neurons in this layer indicating altered neuronal migration due to Tau loss.ConclusionsOur findings suggest the critical involvement of Tau in the neurogenesis suppression of SVZ and OB neurogenic niche under stressful conditions highlighting the role of Tau protein as an essential regulator of stress‐driven plasticity deficits.     

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.     

Competing interactions stabilize pro- and anti-aggregant conformations of human Tau

Aggregation of Tau into amyloid-like fibrils is a key process in neurodegenerative diseases such as Alzheimer. To understand how natively disordered Tau stabilizes conformations that favor pathological aggregation, we applied single-molecule force spectroscopy. Intramolecular interactions that fold polypeptide stretches of ~19 and ~42 amino acids in the functionally important repeat domain of full-length human Tau (hTau40) support aggregation. In contrast, the unstructured N terminus randomly folds long polypeptide stretches >100 amino acids that prevent aggregation. The pro-aggregant mutant hTau40ΔK280 observed in frontotemporal dementia favored the folding of short polypeptide stretches and suppressed the folding of long ones. This trend was reversed in the anti-aggregant mutant hTau40ΔK280/PP. The aggregation inducer heparin introduced strong interactions in hTau40 and hTau40ΔK280 that stabilized aggregation-prone conformations. We show that the conformation and aggregation of Tau are regulated through a complex balance of different intra- and intermolecular interactions.     

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.     

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.     

Ser422 phosphorylation blocks human Tau cleavage by caspase-3: Biochemical implications to Alzheimer’s Disease

Proteolytic truncation of microtubule associated human (h) Tau protein by caspase-3 at the carboxy (C) terminus has been linked to the pathogenesis of Alzheimer’s Disease (AD). This cleavage likely occurs between Asp⁴²¹↓Ser⁴²² leading to the formation of 421-mer truncated Tau protein which has been found to be present as aggregate in high level after phosphorylation in mortal AD brain tissue compared to normal. At least 50 phosphorylation sites involving Ser, Thr and Tyr residues have been identified or proposed in hTau and a selected number of them have been implicated in hTau aggregation following latter’s proteolytic truncation. Interestingly, it is further noted that Ser⁴²² residue present in the P1′ position of hTau caspase-3 cleavage region is a potential phosphorylation site. So we became interested to examine in vitro the effect of phospho-Ser⁴²² residue on hTau cleavage by caspase-3 which is a crucial upstream event associated with hTau self-assembly leading to AD pathogenesis. The goal of this project is to study in vitro the caspase-3 cleavage site of hTau protein and to examine the kinetics of this cleavage following Ser⁴²² phosphorylation and treatment with caspase-3 inhibitors. This is achieved by designing peptides from the sequence of hTau protein containing the proposed caspase-3 cleavage region. Peptides were designed from 441-mer major human Tau protein sequence that encompasses the proposed caspase-3 cleavage site [Asp⁴²¹↓Ser⁴²²]. Corresponding phospho-, dextro-Ser⁴²² and dextro-Asp⁴²¹ analogs were also designed. Peptides were synthesized by solid phase chemistry, purified and fully characterized by mass spectrometry. These were then incubated with recombinant caspase-3 enzyme under identical condition for digestion and analyzed for cleavage by mass spectrometry and RP-HPLC chromatograms. Our results indicated that while the control peptide is efficiently cleaved by caspase-3 at Asp⁴²¹↓Ser⁴²² site producing the expected N- and C-terminal fragment peptides, the corresponding phospho-Ser⁴²² peptide remained completely resistant to the cleavage. Substitution of Asp⁴²¹ by its dextro isoform also blocks peptide cleavage by caspase-3. However substitution of Ser⁴²² by its dextro isoform in the peptide did not affect the cleavage significantly. The above results were further confirmed by caspase-3 digestion experiment in the presence of varying amounts of caspase-3 inhibitor (Ac-DQVD-aldehyde) which was found to block this cleavage in a highly effective manner. Our results highlighted the crucial significance of Ser⁴²² phosphorylation and suggest that the kinase associated with this Ser-phosphorylation may protect Tau from aggregation. Thus specific promoters/activators of this kinase may find useful therapeutic benefits in arresting Tau truncation by caspase-3 and the progression of AD. In addition our data demonstrated that Tau-peptides where Ser⁴²² or Asp⁴²¹ are substituted by their respective dextro isomers, exhibit different cleavage kinetics by caspase-3 and this may have important implications in therapeutic intervention of Tau aggregation and associated AD.     

Tau Trimers Are the Minimal Propagation Unit Spontaneously Internalized to Seed Intracellular Aggregation

Tau amyloid assemblies propagate aggregation from the outside to the inside of a cell, which may mediate progression of the tauopathies. The critical size of Tau assemblies, or “seeds,” responsible for this activity is currently unknown, but this could be important for the design of effective therapies. We studied recombinant Tau repeat domain (RD) and Tau assemblies purified from Alzheimer disease (AD) brain composed largely of full-length Tau. Large RD fibrils were first sonicated to create a range of assembly sizes. We confirmed our ability to resolve stable assemblies ranging from n = 1 to >100 units of Tau using size exclusion chromatography, fluorescence correlation spectroscopy, cross-linking followed by Western blot, and mass spectrometry. All recombinant Tau assemblies bound heparan sulfate proteoglycans on the cell surface, which are required for Tau uptake and seeding, because they were equivalently sensitive to inhibition by heparin and chlorate. However, cells only internalized RD assemblies of n ≥ 3 units. We next analyzed Tau assemblies from AD or control brains. AD brains contained aggregated species, whereas normal brains had predominantly monomer, and no evidence of large assemblies. HEK293 cells and primary neurons spontaneously internalized Tau of n ≥ 3 units from AD brain in a heparin- and chlorate-sensitive manner. Only n ≥ 3-unit assemblies from AD brain spontaneously seeded intracellular Tau aggregation in HEK293 cells. These results indicate that a clear minimum size (n = 3) of Tau seed exists for spontaneous propagation of Tau aggregation from the outside to the inside of a cell, whereas many larger sizes of soluble aggregates trigger uptake and seeding.     

Somatodendritic accumulation of Tau in Alzheimer's disease is promoted by Fyn‐mediated local protein translation

The cause of protein accumulation in neurodegenerative disease is incompletely understood. In Alzheimer's disease (AD), the axonally enriched protein Tau forms hyperphosphorylated aggregates in the somatodendritic domain. Consequently, a process of subcellular relocalization driven by Tau phosphorylation and detachment from microtubules has been proposed. Here, we reveal an alternative mechanism of de novo protein synthesis of Tau and its hyperphosphorylation in the somatodendritic domain, induced by oligomeric amyloid‐β (Aβ) and mediated by the kinase Fyn that activates the ERK/S6 signaling pathway. Activation of this pathway is demonstrated in a range of cellular systems, and in vivo in brains from Aβ‐depositing, Aβ‐injected, and Fyn‐overexpressing mice with Tau accumulation. Both pharmacological inhibition and genetic deletion of Fyn abolish the Aβ‐induced Tau overexpression via ERK/S6 suppression. Together, these findings present a more cogent mechanism of Tau aggregation in disease. They identify a prominent role for neuronal Fyn in integrating signal transduction pathways that lead to the somatodendritic accumulation of Tau in AD.     

Neuronal NLRP1 inflammasome activation of Caspase-1 coordinately regulates inflammatory interleukin-1-beta production and axonal degeneration-associated Caspase-6 activation

Neuronal active Caspase-6 (Casp6) is associated with Alzheimer disease (AD), cognitive impairment, and axonal degeneration. Caspase-1 (Casp1) can activate Casp6 but the expression and functionality of Casp1-activating inflammasomes has not been well-defined in human neurons. Here, we show that primary cultures of human CNS neurons expressed functional Nod-like receptor protein 1 (NLRP1), absent in melanoma 2, and ICE protease activating factor, but not the NLRP3, inflammasome receptor components. NLRP1 neutralizing antibodies in a cell-free system, and NLRP1 siRNAs in neurons hampered stress-induced Casp1 activation. NLRP1 and Casp1 siRNAs also abolished stress-induced Casp6 activation in neurons. The functionality of the NLRP1 inflammasome in serum-deprived neurons was also demonstrated by NLRP1 siRNA-mediated inhibition of speck formation of the apoptosis-associated speck-like protein containing a caspase recruitment domain conjugated to green fluorescent protein. These results indicated a novel stress-induced intraneuronal NLRP1/Casp1/Casp6 pathway. Lipopolysaccharide induced Casp1 and Casp6 activation in wild-type mice brain cortex, but not in that of Nlrp1^−/− and Casp1^−/− mice. NLRP1 immunopositive neurons were increased 25- to 30-fold in AD brains compared with non-AD brains. NLRP1 immunoreactivity in these neurons co-localized with Casp6 activity. Furthermore, the NLRP1/Casp1/Casp6 pathway increased amyloid beta peptide 42 ratio in serum-deprived neurons. Therefore, CNS human neurons express functional NLRP1 inflammasomes, which activate Casp1 and subsequently Casp6, thus revealing a fundamental mechanism linking intraneuronal inflammasome activation to Casp1-generated interleukin-1-β-mediated neuroinflammation and Casp6-mediated axonal degeneration.The lack of efficient treatment for Alzheimer disease (AD) is of high social and economical cost and a growing concern with the aging of the world''s population.¹ Therapies eliminating amyloid beta peptide (Aβ) from AD brains have unfortunately failed to stem progressive cognitive decline. These disappointing results have forced scientists to reconsider treatments against AD; some focusing on targeting Aβ earlier in disease, while others attempting to disaggregate the Tau protein in neurofibrillary tangles (NFT). Recently, the association of several immune responsive genes with increased AD risks^{2, 3, 4} have additionally revived interest in a possible etiological role for inflammation in AD.AD brain inflammation is attributed to activated microglia, which remove Aβ, and secrete neurotoxic molecules that induce neurodegeneration. Interleukin-1-beta (IL-1β), a critical component of brain neuroinflammation, is increased in AD brains⁵ and may contribute to AD pathology by increasing amyloid precursor protein (APP) gene expression, Tau hyperphosphorylation and memory impairment.⁶ However, anti-inflammatory therapies have not provided the expected beneficial effect in AD patients,⁷ suggesting that microglial inflammation may be a consequence of AD. Degenerating neurons are renowned initiators of brain inflammatory responses and the loss of synapses remains the best correlative marker of dementia in AD.⁸ This has incited us to study the response of human neurons to stress and to determine whether specific neuronal molecular events were initiated that link axonal degeneration to an inflammatory response.The active cysteinal Caspase-6 protease (Casp6), associated with axonal degeneration,^{9, 10, 11, 12, 13} is highly abundant in NFT, neuropil threads, and neuritic plaques of AD brains.¹⁴ In some aged non-cognitively impaired individuals, Casp6 activity in the entorhinal cortex and CA1 regions of the hippocampus,¹⁵ two areas initially affected by NFT pathology in AD,¹⁶ correlates significantly with lower cognitive performance.¹⁷ The expression of active Casp6 in CA1 pyramidal neurons of mouse brains is sufficient to induce age-dependent cognitive impairment, in the absence of plaques and tangles, which suggests that active Casp6 in AD brains could be a major contributor to axonal degeneration and cognitive decline.¹⁸Despite substantial evidence implicating Casp6 in AD, the pathways leading to Casp6 activation in neurons are unclear. Caspase-1 (Casp1) activates Casp6 in primary cultures of human CNS neurons.¹⁹ Inflammasome multiprotein complexes, constituted of danger sensing nucleotide-binding oligomerization domain-like receptors or the DNA sensing absent in melanoma 2 (AIM2) component, and the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), recruit and induce Casp1 self-activation.^{20, 21} Functional Nod-like receptor protein 1 (NLRP1), Nod-like receptor protein 3 (NLRP3), AIM2, and ICE protease activating factor (IPAF-1) inflammasomes have been characterized primarily in peripheral macrophages²² and CNS microglia.^{23, 24} Recently, reports have indicated inflammasome receptor expression and activation in rodent neurons. Rat cerebellar granule neurons submitted to oxygen and glucose deprivation or reduced potassium levels increased Nlrp1 mRNA levels.^{25, 26} Nuclear Nlrp1 or functional Nlrp1 inflammasome complexes increased in rat cortical neurons after traumatic brain injury, stroke, and glucose-oxygen deprivation insults.^{27, 28, 29, 30, 31} Neuronal Nlrp1 increased in rats submitted to spinal cord or sciatic nerve injury,^{29, 32} and in aging rat hippocampus or ethanol treated hippocampal slice cultures.^{33, 34} Aim2 induced pyroptosis in rat cortical neuron cultures and traumatic brain injury.³⁵ Nlrp1 has been reported in human brain pyramidal neurons³⁶ and inflammasome receptor mRNAs were observed in human neuron cultures and human Rasmussen''s encephalitis.³⁷Here, we assessed which inflammasome could activate Casp1 and subsequently Casp6 in human primary CNS cultures. We determined which inflammasomes were expressed in naive and stressed neurons and used siRNAs and S-100 cell-free extracts treated with specific inflammasome activators, or antibody blockers, to identify the functional inflammasome. We uncovered that the NLRP1, AIM2, and IPAF-1, but not the NLRP3, inflammasomes were expressed and functional in neurons and that the NLRP1 inflammasome was responsible for Casp1 and subsequently Casp6 activation in serum-deprived and benzylated ATP (BzATP)-stressed neurons. NLRP1 was co-localized with Casp6 activity, immunostained 25- to 30-fold more neurons in AD, and increased Aβ₄₂ in serum-deprived neurons. The NLRP1–Casp1–Casp6 pathway was blocked in lipopolysaccharide (LPS)-treated Nlrp1^−/− and Casp1^−/− mice brains. These results reveal a molecular cascade linking neuronal inflammasome-mediated Casp1 activation to Casp6 activation and provide unexpected novel common neuronal therapeutic targets against neuroinflammation, axonal degeneration, and cognitive impairment in AD.     

Protein Disulfide Isomerase Interacts with Tau Protein and Inhibits Its Fibrillization

Background

Tau protein is implicated in the pathogenesis of neurodegenerative disorders such as tauopathies including Alzheimer disease, and Tau fibrillization is thought to be related to neuronal toxicity. Physiological inhibitors of Tau fibrillization hold promise for developing new strategies for treatment of Alzheimer disease. Because protein disulfide isomerase (PDI) is both an enzyme and a chaperone, and implicated in neuroprotection against Alzheimer disease, we want to know whether PDI can prevent Tau fibrillization. In this study, we have investigated the interaction between PDI and Tau protein and the effect of PDI on Tau fibrillization.

Methodology/Principal Findings

As evidenced by co-immunoprecipitation and confocal laser scanning microscopy, human PDI interacts and co-locates with some endogenous human Tau on the endoplasmic reticulum of undifferentiated SH-SY5Y neuroblastoma cells. The results from isothermal titration calorimetry show that one full-length human PDI binds to one full-length human Tau (or human Tau fragment Tau_244–372) monomer with moderate, micromolar affinity at physiological pH and near physiological ionic strength. As revealed by thioflavin T binding assays, Sarkosyl-insoluble SDS-PAGE, and transmission electron microscopy, full-length human PDI remarkably inhibits both steps of nucleation and elongation of Tau_244–372 fibrillization in a concentration-dependent manner. Furthermore, we find that two molecules of the a-domain of human PDI interact with one Tau_244–372 molecule with sub-micromolar affinity, and inhibit both steps of nucleation and elongation of Tau_244–372 fibrillization more strongly than full-length human PDI.

Conclusions/Significance

We demonstrate for the first time that human PDI binds to Tau protein mainly through its thioredoxin-like catalytic domain a, forming a 1∶1 complex and preventing Tau misfolding. Our findings suggest that PDI could act as a physiological inhibitor of Tau fibrillization, and have applications for developing novel strategies for treatment and early diagnosis of Alzheimer disease.     

A Synergic Role of Caspase-6 and Caspase-3 in Tau Truncation at D421 Induced by H2O2

Tau truncation is widely detected in Alzheimer’s disease brain. Caspases activation is suggested to play a significant role in tau truncation at Aspartate 421 (D421) according to their ability to cleave recombinant tau in vitro. Ample evidence has shown that caspase-6 is involved in cognitive impairment and expressed in AD brain. Reactive oxygen species (ROS) can lead to caspase-6 activation and correlate with AD. Here, we transfected human embryonic kidney 293 (HEK 293) cells with Tau 441 plasmid and investigated the role of caspase-6 and caspase-3 in ROS-mediated tau truncation. Our data demonstrated that H₂O₂ induced oxidative stress and increased tau truncation. Caspase-6 and caspase-3 activity also increased in a dose-dependent manner in HEK 293/Tau cells during H₂O₂ insult. When cells were treated with an ROS inhibitor N-acetyl-l-cysteine, tau truncation was significantly suppressed. Compared with H₂O₂ (100 μM)/non-inhibitor group or single-inhibitor groups (z-VEID-fmk, caspase-6 inhibitor or z-DEVD-fmk, and caspase-3 inhibitor), tau truncation induced by H₂O₂ was effectively reduced in the combinative inhibitors group. Similar results were shown when cells were transfected with specific caspase-3 and caspase-6 siRNA. Inhibition of caspase-6 led to decline of caspase-3 activation. Taken together, our results suggest that the combination of caspase-6 and caspase-3 aggravates tau truncation at D421 induced by H₂O₂. Caspase-6 may play an important part in activating caspase-3. Further investigation of how the synergic role of caspase-6 and caspase-3 affects tau truncation may provide new visions for potential AD therapies.     

N-terminal fragments of tau inhibit full-length tau polymerization in vitro

The polymerization of the microtubule-associated protein, tau, into insoluble filaments is a common thread in Alzheimer's disease and in a variety of frontotemporal dementias. The conformational change required for tau to transition from an extended monomeric state to a filamentous state with a high beta-sheet content involves the extreme N-terminus coming into contact with distal portions of the molecule; however, these exact interactions are incompletely understood. Here we report that a construct representing amino acids 1-196 (Tau196), which itself does not polymerize, inhibits polymerization of full-length tau (hTau40) in vitro. In addition, we trace the inhibitory effect of Tau196 to amino acids 18-42 of the construct. We also provide evidence that the N-terminal tau fragments require a specific C-terminal region of tau (residues 392-421) to exert their inhibitory effect. The fragments are most effective at inhibiting polymerization when present during the initial 5 min; they remain in the soluble fraction of the polymerization reaction, and they increase the amount of soluble hTau40. The fragments also reduce the number and average length of filaments that are formed. Taken together, these results suggest that the N-terminal tau fragments inhibit hTau40 polymerization by interacting with a specific C-terminal sequence, thereby stabilizing a soluble conformation of tau.     

Human Tau Isoforms Assemble into Ribbon-like Fibrils That Display Polymorphic Structure and Stability

Fibrous aggregates of Tau protein are characteristic features of Alzheimer disease. We applied high resolution atomic force and EM microscopy to study fibrils assembled from different human Tau isoforms and domains. All fibrils reveal structural polymorphism; the “thin twisted” and “thin smooth” fibrils resemble flat ribbons (cross-section ∼10 × 15 nm) with diverse twist periodicities. “Thick fibrils” show periodicities of ∼65–70 nm and thicknesses of ∼9–18 nm such as routinely reported for “paired helical filaments” but structurally resemble heavily twisted ribbons. Therefore, thin and thick fibrils assembled from different human Tau isoforms challenge current structural models of paired helical filaments. Furthermore, all Tau fibrils reveal axial subperiodicities of ∼17–19 nm and, upon exposure to mechanical stress or hydrophobic surfaces, disassemble into uniform fragments that remain connected by thin thread-like structures (∼2 nm). This hydrophobically induced disassembly is inhibited at enhanced electrolyte concentrations, indicating that the fragments resemble structural building blocks and the fibril integrity depends largely on hydrophobic and electrostatic interactions. Because full-length Tau and repeat domain constructs assemble into fibrils of similar thickness, the “fuzzy coat” of Tau protein termini surrounding the fibril axis is nearly invisible for atomic force microscopy and EM, presumably because of its high flexibility.     

Hsc70 Rapidly Engages Tau after Microtubule Destabilization

The microtubule-associated protein Tau plays a crucial role in regulating the dynamic stability of microtubules during neuronal development and synaptic transmission. In a group of neurodegenerative diseases, such as Alzheimer disease and other tauopathies, conformational changes in Tau are associated with the initial stages of disease pathology. Folding of Tau into the MC1 conformation, where the amino acids at residues 7–9 interact with residues 312–342, is one of the earliest pathological alterations of Tau in Alzheimer disease. The mechanism of this conformational change in Tau and the subsequent effect on function and association to microtubules is largely unknown. Recent work by our group and others suggests that members of the Hsp70 family play a significant role in Tau regulation. Our new findings suggest that heat shock cognate (Hsc) 70 facilitates Tau-mediated microtubule polymerization. The association of Hsc70 with Tau was rapidly enhanced following treatment with microtubule-destabilizing agents. The fate of Tau released from the microtubule was found to be dependent on ATPase activity of Hsc70. Microtubule destabilization also rapidly increased the MC1 folded conformation of Tau. An in vitro assay suggests that Hsc70 facilitates formation of MC1 Tau. However, in a hyperphosphorylating environment, the formation of MC1 was abrogated, but Hsc70 binding to Tau was enhanced. Thus, under normal circumstances, MC1 formation may be a protective conformation facilitated by Hsc70. However, in a diseased environment, Hsc70 may preserve Tau in a more unstructured state, perhaps facilitating its pathogenicity.     

Diaminothiazoles Modify Tau Phosphorylation and Improve the Tauopathy in Mouse Models

Although Tau accumulation is a feature of several neurodegenerative conditions, treatment options for these conditions are nonexistent. Targeting Tau kinases represents a potential therapeutic approach. Small molecules in the diaminothiazole class are potent Tau kinase inhibitors that target CDK5 and GSK3β. Lead compounds from the series have IC₅₀ values toward CDK5/p25 and GSK3β in the low nanomolar range and no observed toxicity in the therapeutic dose range. Neuronal protective effects and decreased PHF-1 immunoreactivity were observed in two animal models, 3×Tg-AD and CK-p25. Treatment nearly eliminated Sarkosyl-insoluble Tau with the most prominent effect on the phosphorylation at Ser-404. Treatment also induced the recovery of memory in a fear conditioning assay. Given the contribution of both CDK5/p25 and GSK3β to Tau phosphorylation, effective treatment of tauopathies may require dual kinase targeting.     

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator,Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

The activity of Cdk5-p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity and perikaryal aggregations of hyperphosphorylated Tau and neurofilaments, pathogenic hallmarks in neurodegenerative diseases, such as Alzheimer disease and amyotrophic lateral sclerosis, respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5-p25 activity and Tau hyperphosphorylation induced by Aβ peptides in vitro, in HEK293 cells, and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5-p25 hyperactivity and/or Aβ induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues Lys²⁴⁵–Ala²⁷⁷. p5 more effectively inhibited Cdk5-p25 activity than did CIP in vitro. In neuron cells, p5 inhibited deregulated Cdk5-p25 activity but had no effect on the activity of endogenous Cdk5-p35 or on any related endogenous cyclin-dependent kinases in HEK293 cells. Specificity of p5 inhibition in cortical neurons may depend on the p10 domain in p35, which is absent in p25. Furthermore, we have demonstrated that p5 reduced Aβ(1–42)-induced Tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.     

Tau/DDX6 interaction increases microRNA activity

Tauopathies, such as Alzheimer's disease, are characterized by intracellular aggregates of insoluble Tau proteins. Originally described as a microtubule binding protein, recent studies demonstrated additional physiological roles for Tau. The fact that a single protein can regulate multiple cellular functions has posed challenge in terms of understanding mechanistic cues behind the pathology. Here, we used tandem-affinity purification methodology coupled to mass spectrometry to identify novel interaction partners. We found that Tau interacts with DDX6, a DEAD box RNA helicase involved in translation repression and mRNA decay as well as in the miRNA pathway. Our results demonstrate that Tau increases the silencing activity of the miRNA let-7a, miR-21 and miR-124 through DDX6. Importantly, Tau mutations (P301S, P301L) found in the inherited tauopathies, frontotemporal dementia and parkinsonism linked to chromosome 17, disrupt Tau/DDX6 interaction and impair gene silencing by let-7a. Altogether, these data demonstrated a new unexpected role for Tau in regulating miRNA activity.     

Myricetin slows liquid–liquid phase separation of Tau and activates ATG5-dependent autophagy to suppress Tau toxicity

Intraneuronal neurofibrillary tangles composed of Tau aggregates have been widely accepted as an important pathological hallmark of Alzheimer''s disease. A current therapeutic avenue for treating Alzheimer''s disease is aimed at inhibiting Tau accumulation with small molecules such as natural flavonoids. Liquid–liquid phase separation (LLPS) of Tau can lead to its aggregation, and Tau aggregates can then be degraded by autophagy. However, it is unclear whether natural flavonoids modulate the formation of phase-separated Tau droplets or promote autophagy and Tau clearance. Here, using confocal microscopy and fluorescence recovery after photobleaching assays, we report that a natural antioxidant flavonoid compound myricetin slows LLPS of full-length human Tau, shifting the equilibrium phase boundary to a higher protein concentration. This natural flavonoid also significantly inhibits pathological phosphorylation and abnormal aggregation of Tau in neuronal cells and blocks mitochondrial damage and apoptosis induced by Tau aggregation. Importantly, using coimmunoprecipitation and Western blotting, we show that treatment of cells with myricetin stabilizes the interaction between Tau and autophagy-related protein 5 (ATG5) to promote clearance of phosphorylated Tau to indirectly limit its aggregation. Consistently, this natural flavonoid inhibits mTOR pathway, activates ATG5-dependent Tau autophagy, and almost completely suppresses Tau toxicity in neuronal cells. Collectively, these results demonstrate how LLPS and abnormal aggregation of Tau are inhibited by natural flavonoids, bridging the gap between Tau LLPS and aggregation in neuronal cells, and also establish that myricetin could act as an ATG5-dependent autophagic activator to ameliorate the pathogenesis of Alzheimer''s disease.     

人参皂甙Rb1减轻冈田酸诱导的大鼠海马神经元Tau蛋白过度磷酸化

为研究人参皂甙Rb1(ginsenoside Rb1)对冈田酸(okadaic acid,OA)诱导的大鼠海马神经元Tau蛋白过度磷酸化的影响及其可能机制,实验随机分为正常组、溶媒对照组、OA模型组和Rb1预处理组。正常组不作任何处理;Rb1预处理组大鼠分别用5、10、20 mg/kg的Rb1预处理,每天一次,共14 d,于第13天向海马背侧注射1.5μl OA[0.483 μl,溶于10% 二甲基亚砜(dimethysulphoxide,DMSO)];OA模型组大鼠于第13天时海马背侧注射OA,溶媒对照组则注射等体积的生理盐水。各组均于第15天收取标本。通过Biescbowski’s染色、免疫组化和Western blot,分别观察大鼠海马神经元胞体和突起内神经原纤维的改变和磷酸化Tau蛋白的表达水平,同时检测蛋白磷酸酯酶2A(protein phosphatase-2A,PP2A)活性以探讨其作用机制。结果显示:(1)OA模型组与溶媒对照组及正常组比较,海马神经元胞体和突起着色较深,染色不均匀;神经元中Thr231和Sei396位点磷酸化的Tau蛋白和总Tau含量增多;PP2A活性则明显下降(P<0.01):(2)Rb1预处理组大鼠海马神经元胞体和突起染色均匀,神经原纤维走行规则;海马神经元中Thr231和Ser396位点磷酸化的Tau蛋白和总Tau 含量较OA模型组减少,而PP2A活性明显增高(P<0.01)。以上观察结果表明,人参皂甙Rb1可以减轻OA诱导的大鼠海马神经元Tau蛋白过度磷酸化,其机制可能与提高PP2A活性有关。