Human Tau Aggregates Are Permissive to Protein Synthesis-Dependent Memory in Drosophila Tauopathy Models.

Tauopathies including Alzheimer's disease, are characterized by progressive cognitive decline, neurodegeneration, and intraneuronal aggregates comprised largely of the axonal protein Tau. It has been unclear whether cognitive deficits are a consequence of aggregate accumulation thought to compromise neuronal health and eventually lead to neurodegeneration. We use the Drosophila tauopathy model and mixed-sex populations to reveal an adult onset pan-neuronal Tau accumulation-dependent decline in learning efficacy and a specific defect in protein synthesis-dependent memory (PSD-M), but not in its protein synthesis-independent variant. We demonstrate that these neuroplasticity defects are reversible on suppression of new transgenic human Tau expression and surprisingly correlate with an increase in Tau aggregates. Inhibition of aggregate formation via acute oral administration of methylene blue results in re-emergence of deficient memory in animals with suppressed human Tau (hTau)0N4R expression. Significantly, aggregate inhibition results in PSD-M deficits in hTau0N3R-expressing animals, which present elevated aggregates and normal memory if untreated with methylene blue. Moreover, methylene blue-dependent hTau0N4R aggregate suppression within adult mushroom body neurons also resulted in emergence of memory deficits. Therefore, deficient PSD-M on human Tau expression in the Drosophila CNS is not a consequence of toxicity and neuronal loss because it is reversible. Furthermore, PSD-M deficits do not result from aggregate accumulation, which appears permissive, if not protective of processes underlying this memory variant.SIGNIFICANCE STATEMENT Intraneuronal Tau aggregate accumulation has been proposed to underlie the cognitive decline and eventual neurotoxicity that characterizes the neurodegenerative dementias known as tauopathies. However, we show in three experimental settings that Tau aggregates in the Drosophila CNS do not impair but rather appear to facilitate processes underlying protein synthesis-dependent memory within affected neurons.

Liposome nanoparticle conjugation and cell penetrating peptide sequences (CPPs) enhance the cellular delivery of the tau aggregation inhibitor RI-AG03.

Given the pathological role of Tau aggregation in Alzheimer's disease (AD), our laboratory previously developed the novel Tau aggregation inhibitor peptide, RI-AG03. As Tau aggregates accumulate intracellularly, it is essential that the peptide can traverse the cell membrane. Here we examine the cellular uptake and intracellular trafficking of RI-AG03, in both a free and liposome-conjugated form. We also characterize the impact of adding the cell-penetrating peptide (CPP) sequences, polyarginine (polyR) or transactivator of transcription (TAT), to RI-AG03. Our data show that liposome conjugation of CPP containing RI-AG03 peptides, with either the polyR or TAT sequence, increased cellular liposome association three-fold. Inhibition of macropinocytosis modestly reduced the uptake of unconjugated and RI-AG03-polyR-linked liposomes, while having no effect on RI-AG03-TAT-conjugated liposome uptake. Further supporting macropinocytosis-mediated internalization, a 'fair' co-localisation of the free and liposome-conjugated RI-AG03-polyR peptide with macropinosomes and lysosomes was observed. Interestingly, we also demonstrate that RI-AG03-polyR detaches from liposomes following cellular uptake, thereby largely evading organellar entrapment. Collectively, our data indicate that direct membrane penetration and macropinocytosis are key routes for the internalization of liposomes conjugated with CPP containing RI-AG03. Our study also demonstrates that peptide-liposomes are suitable nanocarriers for the cellular delivery of RI-AG03, furthering their potential use in targeting Tau pathology in AD.

Neurodegeneration risk factor, EIF2AK3 (PERK), influences tau protein aggregation.

Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms-unfolded protein response and integrated stress response. Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling in vitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation, whereas inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the endoplasmic reticulum luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small-molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology.

Macrocyclic peptides derived from AcPHF6* and AcPHF6 to selectively modulate the Tau aggregation.

Ten macrocyclic peptides, each comprising 14 amino acids, were designed and synthesized based on the Tau aggregation model hexapeptides AcPHF6* and AcPHF6. The design took into account the aggregation tendencies of each residue in AcPHF6* and AcPHF6, their aggregation models, while employing peptide-based structural design principles including N-methylation to promote turns and to block hydrogen bond propagation and elongation of the aggregation chain. NMR analysis supported that all these peptides adopted an antiparallel ß-sheet conformation. Self-aggregation studies characterized the aggregation properties of these peptides, identifying two peptides with the highest (P3) and lowest (P8) aggregation tendencies. In cross-aggregation studies with the parent peptides AcPHF6* and AcPHF6, P3 and P8 were found to promote and reduce aggregation, respectively. Furthermore, P3 and P8 demonstrated an enhancement and diminution effect on the aggregation of K18wt, indicating their capacity to modulate aggregation even at the macromolecular level. Thus, the two simple peptides, P3 and P8 selectively exhibit pro- or anti-aggregation effects on PHF peptides and Tau. This study, has thus developed structurally well-defined non-complex peptides, derived from AcPHF6* and AcPHF6, to modulate Tau aggregation as desired, offering applications in Tau model studies and the development of Tau aggregation inhibitors or promoters.

Biophysical Studies of Amyloid-Binding Fluorophores to Tau AD Core Fibrils Formed without Cofactors.

Tau is an intrinsically disordered protein involved in several neurodegenerative diseases where a common hallmark is the appearance of tau aggregates in the brain. One common approach to elucidate the mechanisms behind the aggregation of tau has been to recapitulate in vitro the self-assembly process in a fast and reproducible manner. While the seeding of tau aggregation is prompted by negatively charged cofactors, the obtained fibrils are morphologically distinct from those found in vivo. The Tau AD core fragment (TADC, tau 306-378) has emerged as a new model and potential solution for the cofactor-free in vitro aggregation of tau. Here, we use TADC to further study this process combining multiple amyloid-detecting fluorophores and fibril bioimaging. We confirmed by transmission electron microscopy that this fragment forms fibrils after quiescent incubation at 37 °C. We then employed a panel of eight amyloid-binding fluorophores to query the formed species by acquiring their emission spectra. The results obtained showed that nearly all dyes detect TADC self-assembled species. However, the successful monitoring of TADC aggregation kinetics was limited to three fluorophores (X-34, Bis-ANS, and pFTAA) which yielded sigmoidal curves but different aggregation half-times, hinting to different species being detected. Altogether, this study highlights the potential of using multiple extrinsic fluorescent probes, alone or in combination, as tools to further clarify mechanisms behind the aggregation of amyloidogenic proteins.

The dual fates of exogenous tau seeds: Lysosomal clearance versus cytoplasmic amplification.

Tau assembly movement from the extracellular to intracellular space may underlie transcellular propagation of neurodegenerative tauopathies. This begins with tau binding to cell surface heparan sulfate proteoglycans, which triggers macropinocytosis. Pathological tau assemblies are proposed then to exit the vesicular compartment as "seeds" for replication in the cytoplasm. Tau uptake is highly efficient, but only ∼1 to 10% of cells that endocytose aggregates exhibit seeding. Consequently, we studied fluorescently tagged full-length (FL) tau fibrils added to native U2OS cells or "biosensor" cells expressing FL tau or repeat domain. FL tau fibrils bound tubulin. Seeds triggered its aggregation in multiple locations simultaneously in the cytoplasm, generally independent of visible exogenous aggregates. Most exogenous tau trafficked to the lysosome, but fluorescence imaging revealed a small percentage that steadily accumulated in the cytosol. Intracellular expression of Gal3-mRuby, which binds intravesicular galactosides and forms puncta upon vesicle rupture, revealed no evidence of vesicle damage following tau exposure, and most seeded cells had no evidence of endolysosome rupture. However, live-cell imaging indicated that cells with pre-existing Gal3-positive puncta were seeded at a slightly higher rate than the general population, suggesting a potential predisposing role for vesicle instability. Clearance of tau seeds occurred rapidly in both vesicular and cytosolic fractions. The lysosome/autophagy inhibitor bafilomycin inhibited vesicular clearance, whereas the proteasome inhibitor MG132 inhibited cytosolic clearance. Tau seeds that enter the cell thus have at least two fates: lysosomal clearance that degrades most tau, and entry into the cytosol, where seeds amplify, and are cleared by the proteasome.

Liquid-liquid phase separation of protein tau: An emerging process in Alzheimer's disease pathogenesis.

Metabolic reactions within cells occur in various isolated compartments with or without borders, the latter being known as membrane-less organelles (MLOs). The MLOs show liquid-like properties and are formed by a process known as liquid-liquid phase separation (LLPS). MLOs contribute to different molecules interactions such as protein-protein, protein-RNA, and RNA-RNA driven by various factors, such as multivalency of intrinsic disorders. MLOs are involved in several cell signaling pathways such as transcription, immune response, and cellular organization. However, disruption of these processes has been found in different pathologies. Recently, it has been demonstrated that protein aggregates, a characteristic of some neurodegenerative diseases, undergo similar phase separation. Tau protein is known as a major neurofibrillary tangles component in Alzheimer's disease (AD). This protein can undergo phase separation to form a MLO known as tau droplet in vitro and in vivo, and this process can be facilitated by several factors, including crowding agents, RNA, and phosphorylation. Tau droplet has been shown to mature into insoluble aggregates suggesting that this process may precede and induce neurodegeneration in AD. Here we review major factors involved in liquid droplet formation within a cell. Additionally, we highlight recent findings concerning tau aggregation following phase separation in AD, along with the potential therapeutic strategies that could be explored in this process against the progression of this pathology.

Photo-Excited Toluidine Blue Disaggregates the Repeat Tau and Modulates End-Binding Protein EB1, Cytoskeletal Structure in Neuronal Cells.

BACKGROUND/AIMS: Alzheimer's disease is a progressive neurological disorder characterized by the intracellular accumulation of Tau protein aggregates. In the present work, we studied the effect of Toluidine Blue and photo-excited Toluidine Blue on the aggregation of repeat Tau using in vitro assays. METHODS: The in vitro experiments were carried out on recombinant repeat Tau which was purified by cation exchange chromatography. The ThS fluorescence analysis was used to study the aggregation kinetics of Tau. CD spectroscopy and electron microscopy were used to study the secondary structure and morphology of Tau respectively. The actin cytoskeleton modulation was studied in Neuro2a cells with help of immunofluorescent microscopy. RESULTS: Results showed that Toluidine Blue efficiently inhibited the formation of higher-order aggregates, which was evidenced by Thioflavin S fluorescence assay, SDS-PAGE, and TEM. Immunofluorescence studies on the cytoskeleton of Neuro2a cells showed that Toluidine Blue and photo-excited Toluidine Blue treatment at a non-toxic concentration of 0.5 µM stimulated the formation of actin-rich lamellipodia and filopodia structures. Tubulin networks were also differentially modulated after the treatment of Toluidine Blue and photo-excited Toluidine Blue. End-binding protein 1 (EB1) levels were observed to increase after Toluidine Blue and photo-excited Toluidine Blue treatment indicating accelerated microtubule polymerization. CONCLUSION: The overall study suggested that Toluidine Blue inhibited the aggregation of soluble Tau and photo-excited Toluidine Blue disaggregated the pre-formed Tau filaments. In our study, TB and PE-TB were observed to be potent against Tau aggregation. We observed a distinctive modulation of actin, tubulin networks, and EB1 levels after TB and PE-TB treatment, which suggested that TB and PE-TB have potency against cytoskeleton deformities.

A 3D human co-culture to model neuron-astrocyte interactions in tauopathies.

BACKGROUND: Intraneuronal tau aggregation is the major pathological hallmark of neurodegenerative tauopathies. It is now generally acknowledged that tau aggregation also affects astrocytes in a cell non-autonomous manner. However, mechanisms involved are unclear, partly because of the lack of models that reflect the situation in the human tauopathy brain. To accurately model neuron-astrocyte interaction in tauopathies, there is a need for a model that contains both human neurons and human astrocytes, intraneuronal tau pathology and mimics the three-dimensional architecture of the brain. RESULTS: Here we established a novel 100-200 µm thick 3D human neuron/astrocyte co-culture model of tau pathology, comprising homogenous populations of hiPSC-derived neurons and primary human astrocytes in microwell format. Using confocal, electron and live microscopy, we validate the procedures by showing that neurons in the 3D co-culture form pre- and postsynapses and display spontaneous calcium transients within 4 weeks. Astrocytes in the 3D co-culture display bipolar and stellate morphologies with extensive processes that ensheath neuronal somas, spatially align with axons and dendrites and can be found perisynaptically. The complex morphology of astrocytes and the interaction with neurons in the 3D co-culture mirrors that in the human brain, indicating the model's potential to study physiological and pathological neuron-astrocyte interaction in vitro. Finally, we successfully implemented a methodology to introduce seed-independent intraneuronal tau aggregation in the 3D co-culture, enabling study of neuron-astrocyte interaction in early tau pathogenesis. CONCLUSIONS: Altogether, these data provide proof-of-concept for the utility of this rapid, miniaturized, and standardized 3D model for cell type-specific manipulations, such as the intraneuronal pathology that is associated with neurodegenerative disorders.

Artificial intelligence assisted identification of potential tau aggregation inhibitors: ligand- and structure-based virtual screening, in silico ADME, and molecular dynamics study.

Alzheimer's disease (AD) is a severe, growing, multifactorial disorder affecting millions of people worldwide characterized by cognitive decline and neurodegeneration. The accumulation of tau protein into paired helical filaments is one of the major pathological hallmarks of AD and has gained the interest of researchers as a potential drug target to treat AD. Lately, Artificial Intelligence (AI) has revolutionized the drug discovery process by speeding it up and reducing the overall cost. As a part of our continuous effort to identify potential tau aggregation inhibitors, and leveraging the power of AI, in this study, we used a fully automated AI-assisted ligand-based virtual screening tool, PyRMD to screen a library of 12 million compounds from the ZINC database to identify potential tau aggregation inhibitors. The preliminary hits from virtual screening were filtered for similar compounds and pan-assay interference compounds (the compounds containing reactive functional groups which can interfere with the assays) using RDKit. Further, the selected compounds were prioritized based on their molecular docking score with the binding pocket of tau where the binding pockets were identified using replica exchange molecular dynamics simulation. Thirty-three compounds showing good docking scores for all the tau clusters were selected and were further subjected to in silico pharmacokinetic prediction. Finally, top 10 compounds were selected for molecular dynamics simulation and MMPBSA binding free energy calculations resulting in the identification of UNK_175, UNK_1027, UNK_1172, UNK_1173, UNK_1237, UNK_1518, and UNK_2181 as potential tau aggregation inhibitors.