Super-Resolution Imaging Uncovers Nanoscale Tau Aggregate Hyperphosphorylation Patterns in Human Alzheimer's Disease Brain Tissue.

Tau aggregation plays a critical role in Alzheimer's Disease (AD), where tau neurofibrillary tangles (NFTs) are a key pathological hallmark. While much attention has been given to NFTs, emerging evidence underscores nano-sized pre-NFT tau aggregates as potentially toxic entities in AD. By leveraging DNA-PAINT super-resolution microscopy, we visualized and quantified nanoscale tau aggregates (nano-aggregates) in human postmortem brain tissues from intermediate and advanced AD, and Primary Age-Related Tauopathy (PART). Nano-aggregates were predominant across cases, with AD exhibiting a higher burden compared to PART. Hyperphosphorylated tau residues (p-T231, p-T181, and p-S202/T205) were present within nano-aggregates across all AD Braak stages and PART. Moreover, nano-aggregates displayed morphological differences between PART and AD, and exhibited distinct hyperphosphorylation patterns in advanced AD. These findings suggest that changes in nano-aggregate morphology and hyperphosphorylation patterns may exacerbate tau aggregation and AD progression. The ability to detect and profile nanoscale tau aggregates in human brain tissue opens new avenues for studying the molecular underpinnings of tauopathies.

Technological advances in super-resolution microscopy to study cellular processes.

Since its initial demonstration in 2000, far-field super-resolution light microscopy has undergone tremendous technological developments. In parallel, these developments have opened a new window into visualizing the inner life of cells at unprecedented levels of detail. Here, we review the technical details behind the most common implementations of super-resolution microscopy and highlight some of the recent, promising advances in this field.

Tau forms oligomeric complexes on microtubules that are distinct from tau aggregates.

Tau is a microtubule-associated protein, which promotes neuronal microtubule assembly and stability. Accumulation of tau into insoluble aggregates known as neurofibrillary tangles (NFTs) is a pathological hallmark of several neurodegenerative diseases. The current hypothesis is that small, soluble oligomeric tau species preceding NFT formation cause toxicity. However, thus far, visualizing the spatial distribution of tau monomers and oligomers inside cells under physiological or pathological conditions has not been possible. Here, using single-molecule localization microscopy, we show that tau forms small oligomers on microtubules ex vivo. These oligomers are distinct from those found in cells exhibiting tau aggregation and could be precursors of aggregated tau in pathology. Furthermore, using an unsupervised shape classification algorithm that we developed, we show that different tau phosphorylation states are associated with distinct tau aggregate species. Our work elucidates tau's nanoscale composition under nonaggregated and aggregated conditions ex vivo.

In situ analysis and imaging of aromatic amidine at varying ligand densities in solid phase.

We report the development of a fast and accurate fluorescence-based assay for amidine linked to cellulose membranes and Sepharose gel. The assay is founded on the glyoxal reaction, which involves reaction of an amidine group with glyoxal and an aromatic aldehyde, leading to the formation of a fluorophore that can be analyzed and quantified by fluorescence spectroscopy and imaging. While the assay has been reported previously for aromatic amidine estimation in solution phase, here we describe its adaptation and application to amidine linked to diverse forms of solid matrices, particularly benzamidine Sepharose and benzamidine-linked cellulose membranes. These functionalized porous matrices find important application in purification of serine proteases. The efficacy of a protein separation device is determined by, among other factors, the ligand (amidine) density. Hence, a sensitive and reproducible method for amidine quantitation in solid phase is needed. The glyoxal reaction was carried out on microbead-sized Sepharose gel and cellulose membranes. Calibration curves were developed for each phase, which established linearity in the range of 0-0.45 µmol per mL amidine for free amidine in solution, 0-0.45 µmol amidine per mL Sepharose gel, and 0-0.48 µmol per mL cellulose membrane. The assay showed high accuracy (~ 3.4% error), precision (RSD < 2%), and reproducibility. Finally, we show how this fluorescent labeling (glyoxal) method can provide a tool for imaging membranes and ligand distribution through confocal laser scanning microscopy. Graphical abstract.

Modulation of Cu2+ Binding to Sphingosine-1-Phosphate by Lipid Charge.

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that is thought to participate in the regulation of many physiological processes and may play a key role in several diseases. Herein, we found that Cu2+ binds tightly to supported lipid bilayers (SLBs) containing S1P. Specifically, we demonstrated via fluorescence assays that Cu2+-S1P binding was bivalent and sensitive to the concentration of S1P in the SLB. In fact, the apparent equilibrium dissociation constant, KDApp, tightened by a factor of 132 from 4.5 µM to 34 nM as the S1P density was increased from 5.0 to 20 mol %. A major driving force for this apparent tightening was the more negative surface potential with increasing S1P concentration. This potential remained unaltered upon Cu2+ binding at pH 7.4 because two protons were released for every Cu2+ that bound. At pH 5.4, however, Cu2+ could not outcompete protons for the amine and no binding occurred. Moreover, at pH 9.4, the amine was partially deprotonated before Cu2+ binding and the surface potential became more positive on binding. The results for Cu2+-S1P binding were reminiscent of those for Cu2+-phosphatidylserine binding, where a carboxylate group helped to deprotonate the amine. In the case of S1P, however, the phosphate needed to bear two negative charges to facilitate amine deprotonation in the presence of Cu2+.