Tau oligomers are toxic to synapses but not lethal to cells.

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 (Tau RD) 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 β-strand distance (0.47 nm), this points to a potential steric clash. Hence, we investigated the influence of the GFP tag on Tau FL or Tau RD aggregation. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of Tau RD-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 Tau RD filaments.

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 β‐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.

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.

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 β-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.

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 (F3, residues 258-360) harboring the ΔK280 mutation and two proline substitutions (IP & IP) 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.

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.

Abbreviations: AD Alzheimer disease AFM atomic force microscopy CNS central nervous system FRET fluorescence resonance energy transfer GFP green fluorescent protein PHF paired helical filament Tau FL full-length Tau protein, largest isoform in human CNS (Uniprot P10636-F, htau40) Tau FLΔK full-length Tau protein with pro-aggregant mutation ΔK280 Tau RD Tau repeat domain (amyloidogenic domain) Tau RDΔK Tau repeat domain with pro-aggregant mutation ΔK280 Sf9 cell line from Spodoptera frugiperda STEM scanning transmission electron microscopy EM electron microscopy MPL mass per length Wt wildtype AbstractTau 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 proposed for prion pathogenesis. In cell cultures, the process is usually monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (Tau RD , with pro-aggregant mutation, e.g., ΔK280 or P301L, ~13.5 kDa) fused to GFP-based FRET pairs (YFP or CFP, ~28 kD). Since the diameter of the reporter GFP (~3 nm) is ~6.5 times larger than the β-strand distance (0.47nm), this points to a potential steric clash. Hence, we investigated the influence of GFP tagged (N-or C-terminally) Tau RD and Tau FL (full-length Tau) on their aggregation behavior in vitro. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanningtransmission electron microscopy (STEM)), we found that the assembly of Tau RDΔK -GFP was severely inhibited, even in the presence of nucleation enhancers (heparin and/or pre-formed PHFs from Tau RDΔK ). Some rare fiber-like particles had a very different subunit packing from proper PHFs, as judged by STEM. The mass per length (MPL) values of Tau RDΔK fibrils are equivalent to 4.45 molecules/nm, close to the expected value for a paired-helical fiber with 2 protofilaments and cross-β structure. By contrast, the elongated particles formed by Tau RDΔK -GFP have MPL values around ~2, less than half of the values expected for PHFs, indicating that the subunit packing is distinct. Thus, both kinetic and structural observations are incompatible with a model whereby external Tau can form a template for PHF assembly of Tau-GFP in recipient cells. As a consequence, the observed local increase of FRET in recipient cells must be caused by other signalling processes.