Discovery of Orally Available and Brain Penetrant AEP Inhibitors.

Alzheimer's Disease (AD) is the most widespread form of dementia, with one of the pathological hallmarks being the formation of neurofibrillary tangles (NFTs). These tangles consist of phosphorylated Tau fragments. Asparagine endopeptidase (AEP) is a key Tau cleaving enzyme that generates aggregation-prone Tau fragments. Inhibition of AEP to reduce the level of toxic Tau fragment formation could represent a promising therapeutic strategy. Here, we report the first orthosteric, selective, orally bioavailable, and brain penetrant inhibitors with an irreversible binding mode. We outline the development of the series starting from reversible molecules and demonstrate the link between inhibition of AEP and reduction of Tau N368 fragment both in vitro and in vivo.

Spreading of Tau Protein Does Not Depend on Aggregation Propensity.

The stereotypical progression of Tau pathology during Alzheimer disease has been attributed to trans-neuronal spreading of misfolded Tau proteins, followed by prion-like templated aggregation of Tau. The nature of Tau and the cellular mechanisms of Tau spreading are still under debate. We hypothesized that Tau's propensity for aggregation would correlate with its ability to spread across synapses and propagate pathology. To study the progressive propagation of Tau proteins in brain regions relevant for Alzheimer disease, we used mice expressing near-physiological levels of full-length human Tau protein carrying pro-aggregant (TauΔK280, TauΔK) or anti-aggregant (TauΔK280-PP, TauΔK-PP) mutations in the entorhinal cortex (EC). To enhance Tau expression in the EC, we performed EC injections of adeno-associated virus (AAV) particles encoding TauΔK or TauΔK-PP. The brains of injected and non-injected EC/TauΔK and EC/TauΔK-PP mice were studied by immunohistological and biochemical techniques to detect Tau propagation to dentate gyrus (DG) neurons and Tau-induced pathological changes. Pro- and anti-aggregant mice had comparable low transgene expression (~0.2 times endogenous mouse Tau). They accumulated human Tau at similar rates and only in expressing EC neurons, including their axonal projections of the perforant path and presynaptic terminals in the molecular layer of the DG. Pro-aggregant EC/TauΔK mice showed misfolded Tau and synaptic protein alterations in EC neurons, not observed in anti-aggregant EC/TauΔK-PP mice. Additional AAV-mediated expression of TauΔK or TauΔK-PP in EC/TauΔK or EC/TauΔK-PP mice, respectively, increased the human Tau expression to ~0.65 times endogenous mouse Tau, with comparable spreading of TauΔK and TauΔK-PP throughout the EC. There was a low level of transcellular propagation of Tau protein, without pathological phosphorylation or misfolding, as judged by diagnostic antibodies. Additionally, TauΔK but not TauΔK-PP expression induced hippocampal astrogliosis. Low levels of pro- or anti-aggregant full-length Tau show equivalent distributions in EC neurons, independent of their aggregation propensity. Increasing the expression via AAV induce local Tau misfolding in the EC neurons, synaptotoxicity, and astrogliosis and lead to a low level of detectable trans-neuronal spreading of Tau. This depends on its concentration in the EC, but, contrary to expectations, does not depend on Tau's aggregation propensity/misfolding and does not lead to templated misfolding in recipient neurons.

Reversal of Tau-Dependent Cognitive Decay by Blocking Adenosine A1 Receptors: Comparison of Transgenic Mouse Models with Different Levels of Tauopathy.

The accumulation of tau is a hallmark of several neurodegenerative diseases and is associated with neuronal hypoactivity and presynaptic dysfunction. Oral administration of the adenosine A1 receptor antagonist rolofylline (KW-3902) has previously been shown to reverse spatial memory deficits and to normalize the basic synaptic transmission in a mouse line expressing full-length pro-aggregant tau (TauΔK) at low levels, with late onset of disease. However, the efficacy of treatment remained to be explored for cases of more aggressive tauopathy. Using a combination of behavioral assays, imaging with several PET-tracers, and analysis of brain tissue, we compared the curative reversal of tau pathology by blocking adenosine A1 receptors in three mouse models expressing different types and levels of tau and tau mutants. We show through positron emission tomography using the tracer [18F]CPFPX (a selective A1 receptor ligand) that intravenous injection of rolofylline effectively blocks A1 receptors in the brain. Moreover, when administered to TauΔK mice, rolofylline can reverse tau pathology and synaptic decay. The beneficial effects are also observed in a line with more aggressive tau pathology, expressing the amyloidogenic repeat domain of tau (TauRDΔK) with higher aggregation propensity. Both models develop a progressive tau pathology with missorting, phosphorylation, accumulation of tau, loss of synapses, and cognitive decline. TauRDΔK causes pronounced neurofibrillary tangle assembly concomitant with neuronal death, whereas TauΔK accumulates only to tau pretangles without overt neuronal loss. A third model tested, the rTg4510 line, has a high expression of mutant TauP301L and hence a very aggressive phenotype starting at ~3 months of age. This line failed to reverse pathology upon rolofylline treatment, consistent with a higher accumulation of tau-specific PET tracers and inflammation. In conclusion, blocking adenosine A1 receptors by rolofylline can reverse pathology if the pathological potential of tau remains below a threshold value that depends on concentration and aggregation propensity.

Microfluidic Chamber Technology to Study Missorting and Spreading of Tau Protein in Alzheimer's Disease.

Tau is a microtubule-associated protein found mainly in the axons of neurons in the brain. Abnormal changes in Tau (e.g., aggregation, hyperphosphorylation) are hallmarks of Alzheimer's disease. Two processes of relocalization of Tau may be related to early states of the pathology and have received much attention: (1) the redistribution of Tau within cells (termed "somatodendritic missorting") and (2) the release and reuptake of Tau from donor to acceptor cells (termed "spreading"). Because of the tripartite nature of neurons (cell body, dendrites, axons), these changes can be studied by microfluidic chambers (MFCs) which allow separation and observation of Tau in neuronal compartments. In this chapter, we present some methods and research results obtained by using microfluidic devices.

Molecular crowding and RNA synergize to promote phase separation, microtubule interaction, and seeding of Tau condensates.

Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate-mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate-like density for nuclear-envelope Tau. These findings suggest that a complex interplay between interaction partners, post-translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding-competent Tau and lead to distinct cellular Tau accumulations.

A novel D-amino acid peptide with therapeutic potential (ISAD1) inhibits aggregation of neurotoxic disease-relevant mutant Tau and prevents Tau toxicity in vitro.

BACKGROUND: Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder that mainly affects older adults. One of the pathological hallmarks of AD is abnormally aggregated Tau protein that forms fibrillar deposits in the brain. In AD, Tau pathology correlates strongly with clinical symptoms, cognitive dysfunction, and neuronal death. METHODS: We aimed to develop novel therapeutic D-amino acid peptides as Tau fibrillization inhibitors. It has been previously demonstrated that D-amino acid peptides are protease stable and less immunogenic than L-peptides, and these characteristics may render them suitable for in vivo applications. Using a phage display procedure against wild type full-length Tau (TauFL), we selected a novel Tau binding L-peptide and synthesized its D-amino acid version ISAD1 and its retro inversed form, ISAD1rev, respectively. RESULTS: While ISAD1rev inhibited Tau aggregation only moderately, ISAD1 bound to Tau in the aggregation-prone PHF6 region and inhibited fibrillization of TauFL, disease-associated mutant full-length Tau (TauFLΔK, TauFL-A152T, TauFL-P301L), and pro-aggregant repeat domain Tau mutant (TauRDΔK). ISAD1 and ISAD1rev induced the formation of large high molecular weight TauFL and TauRDΔK oligomers that lack proper Thioflavin-positive ß-sheet conformation even at lower concentrations. In silico modeling of ISAD1 Tau interaction at the PHF6 site revealed a binding mode similar to those known for other PHF6 binding peptides. Cell culture experiments demonstrated that ISAD1 and its inverse form are taken up by N2a-TauRDΔK cells efficiently and prevent cytotoxicity of externally added Tau fibrils as well as of internally expressed TauRDΔK. CONCLUSIONS: ISAD1 and related peptides may be suitable for therapy development of AD by promoting off-pathway assembly of Tau, thus preventing its toxicity.

Potent Tau Aggregation Inhibitor D-Peptides Selected against Tau-Repeat 2 Using Mirror Image Phage Display.

Alzheimer's disease and other Tauopathies are associated with neurofibrillary tangles composed of Tau protein, as well as toxic Tau oligomers. Therefore, inhibitors of pathological Tau aggregation are potentially useful candidates for future therapies targeting Tauopathies. Two hexapeptides within Tau, designated PHF6* (275-VQIINK-280) and PHF6 (306-VQIVYK-311), are known to promote Tau aggregation. Recently, the PHF6* segment has been described as the more potent driver of Tau aggregation. We therefore employed mirror-image phage display with a large peptide library to identify PHF6* fibril binding peptides consisting of D-enantiomeric amino acids. The suitability of D-enantiomeric peptides for in vivo applications, which are protease stable and less immunogenic than L-peptides, has already been demonstrated. The identified D-enantiomeric peptide MMD3 and its retro-inverso form, designated MMD3rev, inhibited in vitro fibrillization of the PHF6* peptide, the repeat domain of Tau as well as full-length Tau. Dynamic light scattering, pelleting assays and atomic force microscopy demonstrated that MMD3 prevents the formation of tau ß-sheet-rich fibrils by diverting Tau into large amorphous aggregates. NMR data suggest that the D-enantiomeric peptides bound to Tau monomers with rather low affinity, but ELISA (enzyme-linked immunosorbent assay) data demonstrated binding to PHF6* and full length Tau fibrils. In addition, molecular insight into the binding mode of MMD3 to PHF6* fibrils were gained by in silico modelling. The identified PHF6*-targeting peptides were able to penetrate cells. The study establishes PHF6* fibril binding peptides consisting of D-enantiomeric amino acids as potential molecules for therapeutic and diagnostic applications in AD research.

Novel antibody against low-n oligomers of tau protein promotes clearance of tau in cells via lysosomes.

INTRODUCTION: Tau, a natively unfolded soluble protein, forms abnormal oligomers and insoluble filaments in several neurodegenerative diseases, including Alzheimer disease (AD). Tau-induced toxicity is mainly due to oligomers rather than monomers or fibrils. METHODS: We have developed monoclonal antibodies against purified low-n tau oligomers of the tau repeat domain as a tool to neutralize tau aggregation and toxicity. In vitro aggregation inhibition was tested by thioflavin S, dynamic light scattering (DLS), and atomic force microscopy (AFM). Using a split-luciferase complementation assay and fluorescence-activated cell sorting (FACS), the inhibition of aggregation was analyzed in an N2a cell model of tauopathy. RESULTS: Antibodies inhibited tau aggregation in vitro up to ~90% by blocking tau at an oligomeric state. Some antibodies were able to block tau dimerization/oligomerization in cells, as measured by a split-luciferase complementation assay. Antibodies applied extracellularly were internalized and led to sequestration of tau into lysosomes for degradation. DISCUSSION: Novel low-n tau oligomer specific monoclonal antibody inhibits Tau oligomerization in cells and promotes toxic tau clearance.

A combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications.

Abnormal changes of neuronal Tau protein, such as phosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer's disease. Abnormal phosphorylation is thought to precede aggregation and therefore to promote aggregation, but the nature and extent of phosphorylation remain ill-defined. Tau contains ∼85 potential phosphorylation sites, which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, methodological limitations (e.g. in MS of phosphopeptides, or antibodies against phosphoepitopes) led to conflicting results regarding the extent of Tau phosphorylation in cells. Here we present results from a new approach based on native MS of intact Tau expressed in eukaryotic cells (Sf9). The extent of phosphorylation is heterogeneous, up to ∼20 phosphates per molecule distributed over 51 sites. The medium phosphorylated fraction Pm showed overall occupancies of ∼8 Pi (± 5) with a bell-shaped distribution; the highly phosphorylated fraction Ph had 14 Pi (± 6). The distribution of sites was highly asymmetric (with 71% of all P-sites in the C-terminal half of Tau). All sites were on Ser or Thr residues, but none were on Tyr. Other known posttranslational modifications were near or below our detection limit (e.g. acetylation, ubiquitination). These findings suggest that normal cellular Tau shows a remarkably high extent of phosphorylation, whereas other modifications are nearly absent. This implies that abnormal phosphorylations at certain sites may not affect the extent of phosphorylation significantly and do not represent hyperphosphorylation. By implication, the pathological aggregation of Tau is not likely a consequence of high phosphorylation.

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments.

Tau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism of nucleated polymerization proposed for prion pathogenesis. In cell cultures, the process is often monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (TauRD) with a pro-aggregant mutation, fused to GFP-based FRET pairs. Since the size of the reporter GFP (barrel of ~ 3 nm × 4 nm) is ~ 7 times larger than the ß-strand distance (0.47 nm), this points to a potential steric clash. Hence, we investigated the influence of the GFP tag on TauFL or TauRD aggregation. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of TauRD-GFP was severely inhibited and incompatible with that of Alzheimer filaments. These observations argue against the hypothesis that the propagation of Tau pathology in AD is caused by the prion-like templated aggregation of Tau protein, transmitted via cell-to-cell spreading of Tau. Thus, even though the observed local increase of FRET in recipient cells may be a valid hallmark of a pathological reaction, our data argue that it is caused by a process distinct from assembly of TauRD filaments.

The Plasma Factor XIII Heterotetrameric Complex Structure: Unexpected Unequal Pairing within a Symmetric Complex.

Factor XIII (FXIII) is a predominant determinant of clot stability, strength, and composition. Plasma FXIII circulates as a pro-transglutaminase with two catalytic A subunits and two carrier-protective B subunits in a heterotetramer (FXIII-A2B2). FXIII-A2 and -B2 subunits are synthesized separately and then assembled in plasma. Following proteolytic activation by thrombin and calcium-mediated dissociation of the B subunits, activated FXIII (FXIIIa) covalently cross links fibrin, promoting clot stability. The zymogen and active states of the FXIII-A subunits have been structurally characterized; however, the structure of FXIII-B subunits and the FXIII-A2B2 complex have remained elusive. Using integrative hybrid approaches including atomic force microscopy, cross-linking mass spectrometry, and computational approaches, we have constructed the first all-atom model of the FXIII-A2B2 complex. We also used molecular dynamics simulations in combination with isothermal titration calorimetry to characterize FXIII-A2B2 assembly, activation, and dissociation. Our data reveal unequal pairing of individual subunit monomers in an otherwise symmetric complex, and suggest this unusual structure is critical for both assembly and activation of this complex. Our findings enhance understanding of mechanisms associating FXIII-A2B2 mutations with disease and have important implications for the rational design of molecules to alter FXIII assembly or activity to reduce bleeding and thrombotic complications.

Disruption of Structural Disulfides of Coagulation FXIII-B Subunit; Functional Implications for a Rare Bleeding Disorder.

Congenital FXIII deficiency is a rare bleeding disorder in which mutations are detected in F13A1 and F13B genes that express the two subunits of coagulation FXIII, the catalytic FXIII-A, and protective FXIII-B. Mutations in FXIII-B subunit are considerably rarer compared to FXIII-A. Three mutations in the F13B gene have been reported on its structural disulfide bonds. In the present study, we investigate the structural and functional importance of all 20 structural disulfide bonds in FXIII-B subunit. All disulfide bonds were ablated by individually mutating one of its contributory cysteine's, and these variants were transiently expressed in HEK293t cell lines. The expression products were studied for stability, secretion, the effect on oligomeric state, and on FXIII-A activation. The structural flexibility of these disulfide bonds was studied using classical MD simulation performed on a FXIII-B subunit monomer model. All 20 FXIII-B were found to be important for the secretion and stability of the protein since ablation of any of these led to a secretion deficit. However, the degree of effect that the disruption of disulfide bond had on the protein differed between individual disulfide bonds reflecting a functional hierarchy/diversity within these disulfide bonds.

Suppressing Tau Aggregation and Toxicity by an Anti-Aggregant Tau Fragment.

Tau aggregation is a hallmark of a group of neurodegenerative diseases termed Tauopathies. Reduction of aggregation-prone Tau has emerged as a promising therapeutic approach. Here, we show that an anti-aggregant Tau fragment (F3ΔKPP, residues 258-360) harboring the ΔK280 mutation and two proline substitutions (I277P & I308P) in the repeat domain can inhibit aggregation of Tau constructs in vitro, in cultured cells and in vivo in a Caenorhabditis elegans model of Tau aggregation. The Tau fragment reduced Tau-dependent cytotoxicity in a N2a cell model, suppressed the Tau-mediated neuronal dysfunction and ameliorated the defective locomotion in C. elegans. In vitro the fragment competes with full-length Tau for polyanionic aggregation inducers and thus inhibits Tau aggregation. Our combined in vitro and in vivo results suggest that the anti-aggregant Tau fragment may potentially be used to address the consequences of Tau aggregation in Tauopathies.

Pathological missorting of endogenous MAPT/Tau in neurons caused by failure of protein degradation systems.

Missorting of MAPT/Tau represents one of the early signs of neurodegeneration in Alzheimer disease. The triggers for this are still a matter of debate. Here we investigated the sorting mechanisms of endogenous MAPT in mature primary neurons using microfluidic chambers (MFCs) where cell compartments can be observed separately. Blocking protein degradation pathways with proteasomal or autophagy inhibitors dramatically increased the missorting of MAPT in dendrites on the neuritic side, suggesting that degradation of MAPT in dendrites is a major determinant for the physiological axonal distribution of MAPT. Such missorted dendritic MAPT differed in its phosphorylation pattern from axonal MAPT. By contrast, enhancing autophagy or proteasomal pathways strongly reduced MAPT missorting, thereby confirming the role of protein degradation pathways in the polar distribution of MAPT. Dendritic missorting of MAPT by blocking protein degradation resulted in the loss of spines but not in overall cell toxicity. Inhibition of local protein synthesis in dendrites eliminated the missorting of MAPT, indicating that the accumulation of dendritic MAPT is locally generated. In support of this, a substantial fraction of Mapt/Tau mRNA was detected in dendrites. Taken together, our results indicate that the autophagy and proteasomal pathways play important roles in fine-tuning dendritic MAPT levels and thereby prevent synaptic toxicity caused by MAPT accumulation. Abbreviations Ani: anisomycin; Baf: bafilomycin A1; BSA: bovine serum albumin; cAMP: cyclic adenosine monophosphate; CHX: cycloheximide; DMSO: dimethyl sulfoxide; DIV: days in vitro; Epo: epoxomicin; E18: embryonic day 18; FISH: fluorescence in situ hybridization; IgG: immunoglobulin; kDa: kilodalton; Lac: lactacystin; LDH: lactate dehydrogenase; MFC: microfluidic chambers; MAPs: microtubule-associated proteins; MAPT/Tau: microtubule-associated protein tau; PVDF: polyvinylidene difluoride; PBS: phosphate-buffered saline; PRKA: protein kinase AMP-activated; RD150: round device 150; RT: room temperature; SDS: sodium dodecyl sulfate; SEM: standard error of the mean; Wor: wortmannin.

Purification and Characterization of Low-n Tau Oligomers.

Deposition of Tau aggregates in patient's brains is a hallmark of several neurodegenerative diseases collectively called Tauopathies. One of the most studied Tauopathies is Alzheimer disease (AD) in which Tau protein aggregates into filaments and coalesces into neurofibrillary tangles. The distribution of Tau filaments is a reliable indicator of the clinical stages of AD (Braak stages), but intermediate oligomeric assemblies of Tau are considered to be more directly toxic to neurons than late stage filaments. Studying the elusive role of Tau oligomers has been difficult because of their dynamic nature and paucity of methods to purify them in vitro. In this chapter, we describe methods to purify Tau oligomers to near homogeneity and to characterize them by hydrophobic interaction chromatography and biophysical methods such as fluorescence spectrophotometry, dynamic light scattering, atomic force microscopy, and others. Functional characterization includes the assessment of synapses and toxicity assays which show that oligomers can damage synapses locally but show little toxicity to neurons globally.

Extracellular low-n oligomers of tau cause selective synaptotoxicity without affecting cell viability.

INTRODUCTION: Tau-mediated toxicity in Alzheimer's disease is thought to operate through low-n oligomers, rather than filamentous aggregates. However, the nature of oligomers and pathways of toxicity are poorly understood. Therefore, we investigated structural and functional aspects of highly purified oligomers of a pro-aggregant tau species. METHODS: Purified oligomers of the tau repeat domain were characterized by biophysical and structural methods. Functional aspects were investigated by cellular assays ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay of cell viability, lactate dehydrogenase release assay [for cell toxicity], reactive oxygen species production, and calcium assay), combined with analysis of neuronal dendritic spines exposed to oligomers. RESULTS: Purified low-n oligomers are roughly globular, with sizes around 1.6 to 5.4 nm, exhibit an altered conformation, but do not have substantial ß-structure. Treatment of primary neurons with oligomers impairs spine morphology and density, accompanied by increased reactive oxygen species and intracellular calcium, but without affecting cell viability (by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay of cell viability and lactate dehydrogenase release assay [for cell toxicity]). DISCUSSION: Tau oligomers are toxic to synapses but not lethal to cells.

The release and trans-synaptic transmission of Tau via exosomes.

BACKGROUND: Tau pathology in AD spreads in a hierarchical pattern, whereby it first appears in the entorhinal cortex, then spreads to the hippocampus and later to the surrounding areas. Based on this sequential appearance, AD can be classified into six stages ("Braak stages"). The mechanisms and agents underlying the progression of Tau pathology are a matter of debate. Emerging evidence indicates that the propagation of Tau pathology may be due to the transmission of Tau protein, but the underlying pathways and Tau species are not well understood. In this study we investigated the question of Tau spreading via small extracellular vesicles called exosomes. METHODS: Exosomes from different sources were analyzed by biochemical methods and electron microscopy (EM) and cryo-EM. Microfluidic devices that allow the culture of cell populations in different compartments were used to investigate the spreading of Tau. RESULTS: We show that Tau protein is released by cultured primary neurons or by N2a cells overexpressing different Tau constructs via exosomes. Neuron-derived exosomal Tau is hypo-phosphorylated, compared with cytosolic Tau. Depolarization of neurons promotes release of Tau-containing exosomes, highlighting the importance of neuronal activity. Using microfluidic devices we show that exosomes mediate trans-neuronal transfer of Tau depending on synaptic connectivity. Tau spreading is achieved by direct transmission of exosomes between neurons. In organotypic hippocampal slices, Tau-containing exosomes in conditioned medium are taken up by neurons and microglia, not astrocytes. In N2a cells, Tau assemblies are released via exosomes. They can induce inclusions of other Tau molecules in N2a cells expressing mutant human Tau. We also studied exosomes from cerebrospinal fluid in AD and control subjects containing monomeric and oligomeric Tau. Split-luciferase complementation reveals that exosomes from CSF can promote Tau aggregation in cultured cells. CONCLUSION: Our study demonstrates that exosomes contribute to trans-synaptic Tau transmission, and thus offer new approches to control the spreading of pathology in AD and other tauopathies.

Stages and conformations of the Tau repeat domain during aggregation and its effect on neuronal toxicity.

Several neurodegenerative diseases are characterized by the aggregation and posttranslational modifications of Tau protein. Its "repeat domain" (TauRD) is mainly responsible for the aggregation properties, and oligomeric forms are thought to dominate the toxic effects of Tau. Here we investigated the conformational transitions of this domain during oligomerization and aggregation in different states of ß-propensity and pseudo-phosphorylation, using several complementary imaging and spectroscopic methods. Although the repeat domain generally aggregates more readily than full-length Tau, its aggregation was greatly slowed down by phosphorylation or pseudo-phosphorylation at the KXGS motifs, concomitant with an extended phase of oligomerization. Analogous effects were observed with pro-aggregant variants of TauRD. Oligomers became most evident in the case of the pro-aggregant mutant TauRDΔK280, as monitored by atomic force microscopy, and the fluorescence lifetime of Alexa-labeled Tau (time-correlated single photon counting (TCSPC)), consistent with its pronounced toxicity in mouse models. In cell models or primary neurons, neither oligomers nor fibrils of TauRD or TauRDΔK280 had a toxic effect, as seen by assays with lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, respectively. However, oligomers of pro-aggregant TauRDΔK280 specifically caused a loss of spine density in differentiated neurons, indicating a locally restricted impairment of function.