Tau seed amplification assay reveals relationship between seeding and pathological forms of tau in Alzheimer's disease brain.

Tau seed amplification assays (SAAs) directly measure the seeding activity of tau and would therefore be ideal biomarkers for clinical trials targeting seeding-competent tau in Alzheimer's disease (AD). However, the precise relationship between tau seeding measured by SAA and the levels of pathological forms of tau in the AD brain remains unknown. We developed a new tau SAA based on full-length 0N3R tau with sensitivity in the low fg/ml range and used it to characterize 103 brain samples from three independent cohorts. Tau seeding clearly discriminated between AD and control brain samples. Interestingly, seeding was absent in Progressive Supranuclear Palsy (PSP) putamen, suggesting that our tau SAA did not amplify 4R tau aggregates from PSP brain. The specificity of our tau SAA for AD brain was further supported by analysis of matched hippocampus and cerebellum samples. While seeding was detected in hippocampus from Braak stages I-II, no seeding was present in AD cerebellum that is devoid of tau inclusions. Analysis of 40 middle frontal gyrus samples encompassing all Braak stages showed that tau SAA seeding activity gradually increased with Braak stage. This relationship between seeding activity and the presence of tau inclusions in AD brain was further supported by robust correlations between tau SAA results and the levels of phosphorylated tau212/214, phosphorylated tau181, aggregated tau, and sarkosyl-insoluble tau. Strikingly, we detected tau seeding in the middle frontal gyrus already at Braak stage II-III, suggesting that tau SAA can detect tau pathology earlier than conventional immunohistochemical staining. In conclusion, our data suggest a quantitative relationship between tau seeding activity and pathological forms of tau in the human brain and provides an important basis for further development of tau SAA for accessible human samples.

High diagnostic performance of independent alpha-synuclein seed amplification assays for detection of early Parkinson's disease.

Alpha-synuclein seed amplification assays (αSyn-SAAs) are promising diagnostic tools for Parkinson's disease (PD) and related synucleinopathies. They enable detection of seeding-competent alpha-synuclein aggregates in living patients and have shown high diagnostic accuracy in several PD and other synucleinopathy patient cohorts. However, there has been confusion about αSyn-SAAs for their methodology, nomenclature, and relative accuracies when performed by various laboratories. We compared αSyn-SAA results obtained from three independent laboratories to evaluate reproducibility across methodological variations. We utilized the Parkinson's Progression Markers Initiative (PPMI) cohort, with DATSCAN data available for comparison, since clinical diagnosis of early de novo PD is critical for neuroprotective trials, which often use dopamine transporter imaging to enrich their cohorts. Blinded cerebrospinal fluid (CSF) samples for a randomly selected subset of PPMI subjects (30 PD, 30 HC, and 20 SWEDD), from both baseline and year 3 collections for the PD and HC groups (140 total CSF samples) were analyzed in parallel by each lab according to their own established and optimized αSyn-SAA protocols. The αSyn-SAA results were remarkably similar across laboratories, displaying high diagnostic performance (sensitivity ranging from 86 to 96% and specificity from 93 to 100%). The assays were also concordant for samples with results that differed from clinical diagnosis, including 2 PD patients determined to be clinically inconsistent with PD at later time points. All three assays also detected 2 SWEDD subjects as αSyn-SAA positive who later developed PD with abnormal DAT-SPECT. These multi-laboratory results confirm the reproducibility and value of αSyn-SAA as diagnostic tools, illustrate reproducibility of the assay in expert hands, and suggest that αSyn-SAA has potential to provide earlier diagnosis with comparable or superior accuracy to existing methods.

N368-Tau fragments generated by legumain are detected only in trace amount in the insoluble Tau aggregates isolated from AD brain.

Intraneuronal insoluble inclusions made of Tau protein are neuropathological hallmarks of Alzheimer Disease (AD). Cleavage of Tau by legumain (LGMN) has been proposed to be crucial for aggregation of Tau into fibrils. However, it remains unclear if LGMN-cleaved Tau fragments accumulate in AD Tau inclusions.Using an in vitro enzymatic assay and non-targeted mass spectrometry, we identified four putative LGMN cleavage sites at Tau residues N167-, N255-, N296- and N368. Cleavage at N368 generates variously sized N368-Tau fragments that are aggregation prone in the Thioflavin T assay in vitro. N368-cleaved Tau is not detected in the brain of legumain knockout mice, indicating that LGMN is required for Tau cleavage in the mouse brain in vivo. Using a targeted mass spectrometry method in combination with tissue fractionation and biochemical analysis, we investigated whether N368-cleaved Tau is differentially produced and aggregated in brain of AD patients and control subjects. In brain soluble extracts, despite reduced uncleaved Tau in AD, levels of N368-cleaved Tau are comparable in AD and control hippocampus, suggesting that LGMN-mediated cleavage of Tau is not altered in AD. Consistently, levels of activated, cleaved LGMN are also similar in AD and control brain extracts. To assess the potential accumulation of N368-cleaved Tau in insoluble Tau aggregates, we analyzed sarkosyl-insoluble extracts from AD and control hippocampus. Both N368-cleaved Tau and uncleaved Tau were significantly increased in AD as a consequence of pathological Tau inclusions accumulation. However, the amount of N368-cleaved Tau represented only a very minor component (< 0.1%) of insoluble Tau.Our data indicate that LGMN physiologically cleaves Tau in the mouse and human brain generating N368-cleaved Tau fragments, which remain largely soluble and are present only in low proportion in Tau insoluble aggregates compared to uncleaved Tau. This suggests that LGMN-cleaved Tau has limited role in the progressive accumulation of Tau inclusions in AD.

Label-free detection of small-molecule binding to a GPCR in the membrane environment.

Evaluation of drug-target interaction kinetics is becoming increasingly important during the drug-discovery process to investigate selectivity of a drug and predict in vivo target occupancy. To date, it remains challenging to obtain kinetic information for interactions between G-protein-coupled receptors (GPCRs) and small-molecule ligands in a label-free manner. Often GPCRs need to be solubilized or even stabilized by mutations which can be difficult and is time consuming. In addition, it is often unclear if the native conformation of the receptors is sustained. In this study, surface plasmon resonance (SPR) and surface acoustic wave (SAW) technologies have been used to detect ligand binding to the GPCR chemokine (C-X-C motif) receptor 4 (CXCR4) expressed in lipoparticles. We first evaluated different strategies to immobilize CXCR4-expressing lipoparticles. The highest small-molecule binding signal in SPR and SAW was achieved with a matrix-free carboxymethylated sensor chip coated with wheat germ agglutinin for lipoparticle capturing. Next, the binding kinetics of the anti-CXCR4 antibody 12G5 raised against a conformational epitope (k(on)=1.83×10(6)M(-1)s(-1), k(off)=2.79×10(-4) s(-1)) and the small molecule AMD3100 (k(on)=5.46×10(5)M(-1)s(-1), k(off)=1.01×10(-2)s(-1)) were assessed by SAW. Our kinetic and affinity data are consistent with previously published radioligand-binding experiments using cells and label-free experiments with solubilized CXCR4. This is the first study demonstrating label-free kinetic characterization of small-molecule binding to a GPCR in the membrane environment. The presented method holds the potential to greatly facilitate label-free assay development for GPRCs that can be expressed at high levels in lipoparticles.

Design strategies to address kinetics of drug binding and residence time.

The kinetics of drug binding and drug residence time are recognized to be important in the clinical effectiveness of drug candidates. In most cases a long residence time of the drug-target complex results in an extended duration of pharmacodynamic activity, even when systemic concentrations of drug have been notably reduced through elimination routes. Hence, if selective for target, long residence times can increase the duration of drug efficacy in vivo and can significantly diminish the potential for off-target-mediated toxicities. Furthermore, a compound with a slower dissociation rate may allow a reduced dosing schedule relative to a compound with a rapid dissociation rate. Factors contributing to long residence time that could be useful to medicinal chemists in the prospective design of compounds with long residence times will be discussed in this perspective. Particular emphasis will be on case studies highlighting how kinetics can be measured, modulated based on supporting structure kinetic relationships and whether these effects are translatable into man.

In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation.

The assembly and composition of ribonucleic acid (RNA)-transporting particles for asymmetric messenger RNA (mRNA) localization is not well understood. During mitosis of budding yeast, the Swi5p-dependent HO expression (SHE) complex transports a set of mRNAs into the daughter cell. We recombinantly reconstituted the core SHE complex and assessed its properties. The cytoplasmic precomplex contains only one motor and is unable to support continuous transport. However, a defined interaction with a second, RNA-bound precomplex after its nuclear export dimerizes the motor and activates processive RNA transport. The run length observed in vitro is compatible with long-distance transport in vivo. Surprisingly, SHE complexes that either contain or lack RNA cargo show similar motility properties, demonstrating that the RNA-binding protein and not its cargo activates motility. We further show that SHE complexes have a defined size but multimerize into variable particles upon binding of RNAs with multiple localization elements. Based on these findings, we provide an estimate of number, size, and composition of such multimeric SHE particles in the cell.

Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs.

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the -3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.