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 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

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

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 (Tau(FL)), 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 Tau(FL), disease-associated mutant full-length Tau (Tau(FL Delta K), Tau(FL-A152T), Tau(FL-P301L)), and pro-aggregant repeat domain Tau mutant (Tau(RD Delta K)). ISAD1 and ISAD1rev induced the formation of large high molecular weight Tau(FL) and Tau(RD Delta K) oligomers that lack proper Thioflavin-positive beta-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-Tau(RD Delta K) cells efficiently and prevent cytotoxicity of externally added Tau fibrils as well as of internally expressed Tau(RD Delta K). Conclusions ISAD1 and related peptides may be suitable for therapy development of AD by promoting off-pathway assembly of Tau, thus preventing its toxicity

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

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

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

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

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

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc