DJ-1 Molecular Chaperone Activity Depresses Tau Aggregation Propensity through Interaction with Monomers.

Tau aggregate-bearing lesions are pathological markers and potential mediators of tauopathic neurodegenerative diseases, including Alzheimer's disease. The molecular chaperone DJ-1 colocalizes with tau pathology in these disorders, but it has been unclear what functional link exists between them. In this study, we examined the consequences of tau/DJ-1 interaction as isolated proteins in vitro. When added to full-length 2N4R tau under aggregation-promoting conditions, DJ-1 inhibited both the rate and extent of filament formation in a concentration-dependent manner. Inhibitory activity was low affinity, did not require ATP, and was not affected by substituting oxidation incompetent missense mutation C106A for wild-type DJ-1. In contrast, missense mutations previously linked to familial Parkinson's disease and loss of α-synuclein chaperone activity, M26I and E64D, displayed diminished tau chaperone activity relative to wild-type DJ-1. Although DJ-1 directly bound the isolated microtubule-binding repeat region of tau protein, exposure of preformed tau seeds to DJ-1 did not diminish seeding activity in a biosensor cell model. These data reveal DJ-1 to be a holdase chaperone capable of engaging tau as a client in addition to α-synuclein. Our findings support a role for DJ-1 as part of an endogenous defense against the aggregation of these intrinsically disordered proteins.

Blood RNA transcripts reveal similar and differential alterations in fundamental cellular processes in Alzheimer's disease and other neurodegenerative diseases.

BACKGROUND: Dysfunctional processes in Alzheimer's disease and other neurodegenerative diseases lead to neural degeneration in the central and peripheral nervous system. Research demonstrates that neurodegeneration of any kind is a systemic disease that may even begin outside of the region vulnerable to the disease. Neurodegenerative diseases are defined by the vulnerabilities and pathology occurring in the regions affected. METHOD: A random forest machine learning analysis on whole blood transcriptomes from six neurodegenerative diseases generated unbiased disease-classifying RNA transcripts subsequently subjected to pathway analysis. RESULTS: We report that transcripts of the blood transcriptome selected for each of the neurodegenerative diseases represent fundamental biological cell processes including transcription regulation, degranulation, immune response, protein synthesis, apoptosis, cytoskeletal components, ubiquitylation/proteasome, and mitochondrial complexes that are also affected in the brain and reveal common themes across six neurodegenerative diseases. CONCLUSION: Neurodegenerative diseases share common dysfunctions in fundamental cellular processes. Identifying regional vulnerabilities will reveal unique disease mechanisms. HIGHLIGHTS: Transcriptomics offer information about dysfunctional processes. Comparing multiple diseases will expose unique malfunctions within diseases. Blood RNA can be used ante mortem to track expression changes in neurodegenerative diseases. Protocol standardization will make public datasets compatible.

Blood Transcript Biomarkers Selected by Machine Learning Algorithm Classify Neurodegenerative Diseases including Alzheimer's Disease.

The clinical diagnosis of neurodegenerative diseases is notoriously inaccurate and current methods are often expensive, time-consuming, or invasive. Simple inexpensive and noninvasive methods of diagnosis could provide valuable support for clinicians when combined with cognitive assessment scores. Biological processes leading to neuropathology progress silently for years and are reflected in both the central nervous system and vascular peripheral system. A blood-based screen to distinguish and classify neurodegenerative diseases is especially interesting having low cost, minimal invasiveness, and accessibility to almost any world clinic. In this study, we set out to discover a small set of blood transcripts that can be used to distinguish healthy individuals from those with Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, or frontotemporal dementia. Using existing public datasets, we developed a machine learning algorithm for application on transcripts present in blood and discovered small sets of transcripts that distinguish a number of neurodegenerative diseases with high sensitivity and specificity. We validated the usefulness of blood RNA transcriptomics for the classification of neurodegenerative diseases. Information about features selected for the classification can direct the development of possible treatment strategies.

A theoretical study of polymorphism in VQIVYK fibrils.

The VQIVYK fragment from the Tau protein, also known as PHF6, is essential for aggregation of Tau into neurofibrillary lesions associated with neurodegenerative diseases. VQIVYK itself forms amyloid fibrils composed of paired ß-sheets. Therefore, the full Tau protein and VQIVYK fibrils have been intensively investigated. A central issue in these studies is polymorphism, the ability of a protein to fold into more than one structure. Using all-atom molecular simulations, we generate five stable polymorphs of VQIVYK fibrils, establish their relative free energy with umbrella sampling methods, and identify the side chain interactions that provide stability. The two most stable polymorphs, which have nearly equal free energy, are formed by interdigitation of the mostly hydrophobic VIY "face" sides of the ß-sheets. Another stable polymorph is formed by interdigitation of the QVK "back" sides. When we turn to examine structures from cryo-electron microscopy experiments on Tau filaments taken from diseased patients or generated in vitro, we find that the pattern of side chain interactions found in the two most stable face-to-face as well as the back-to-back polymorphs are recapitulated in amyloid structures of the full protein. Thus, our studies suggest that the interactions stabilizing PHF6 fibrils explain the amyloidogenicity of the VQIVYK motif within the full Tau protein and provide justification for the use of VQIVYK fibrils as a test bed for the design of molecules that identify or inhibit amyloid structures.

Quantification of Tau Protein Lysine Methylation in Aging and Alzheimer's Disease.

Tau is a microtubule-associated protein that normally interacts in monomeric form with the neuronal cytoskeleton. In Alzheimer's disease, however, it aggregates to form the structural component of neurofibrillary lesions. The transformation is controlled in part by age- and disease-associated post-translational modifications. Recently we reported that tau isolated from cognitively normal human brain was methylated on lysine residues, and that high-stoichiometry methylation depressed tau aggregation propensity in vitro. However, whether methylation stoichiometry reached levels needed to influence aggregation propensity in human brain was unknown. Here we address this problem using liquid chromatography-tandem mass spectrometry approaches and human-derived tau samples. Results revealed that lysine methylation was present in soluble tau isolated from cognitively normal elderly cases at multiple sites that only partially overlapped with the distributions reported for cognitively normal middle aged and AD cohorts, and that the quality of methylation shifted from predominantly dimethyl-lysine to monomethyl-lysine with aging and disease. However, bulk mol methylation/mol tau stoichiometries never exceeded 1 mol methyl group/mol tau protein. We conclude that lysine methylation is a physiological post-translational modification of tau protein that changes qualitatively with aging and disease, and that pharmacological elevation of tau methylation may provide a means for protecting against pathological tau aggregation.

The role of annealing and fragmentation in human tau aggregation dynamics.

Alzheimer's disease pathogenesis is associated with the conversion of monomeric tau protein into filamentous aggregates. Because both toxicity and prion-like spread of pathogenic tau depend in part on aggregate size, the processes that underlie filament formation and size distribution are of special importance. Here, using a combination of biophysical and computational approaches, we investigated the fibrillation dynamics of the human tau isoform 2N4R. We found that tau filaments engage in a previously uncharacterized secondary process involving end-to-end annealing and that rationalization of empirical aggregation data composed of total protomer concentrations and fibril length distributions requires inclusion of this process along with filament fragmentation. We noted that annealing of 2N4R tau filaments is robust, with an intrinsic association rate constant of a magnitude similar to that mediating monomer addition and consistent with diffusion-mediated protein-protein interactions in the absence of long-range attractive forces. In contrast, secondary nucleation on the surface of tau filaments did not detectably contribute to tau aggregation dynamics. These results indicate that tau filament ends engage in a range of homotypic interactions involving monomers, oligomers, and filaments. They further indicate that, in the case of tau protein, fibril annealing and fragmentation along with primary nucleation and elongation are the major processes controlling filament size distribution.

A liquid chromatography tandem mass spectroscopy approach for quantification of protein methylation stoichiometry.

Post-translational modifications are biologically important and wide-spread modulators of protein function. Although methods for detecting the presence of specific modifications are becoming established, approaches for quantifying their mol modification/mol protein stoichiometry are less well developed. Here we introduce a ratiometric, label-free, targeted liquid chromatography tandem mass spectroscopy-based method for estimating Lys and Arg methylation stoichiometry on post-translationally modified proteins. Methylated Lys and Arg were detected with limits of quantification at low fmol and with linearity extending from 20 to 5000 fmol. This level of sensitivity allowed estimation of methylation stoichiometry from microgram quantities of various proteins, including those derived from either recombinant or tissue sources. The method also disaggregated total methylation stoichiometry into its elementary mono-, di-, and tri-methylated residue components. In addition to being compatible with kinetic experiments of protein methylation, the approach will be especially useful for characterizing methylation states of proteins isolated from cells and tissues.

Analyzing Tau Aggregation with Electron Microscopy.

Conversion of monomeric tau protein into filamentous aggregates is a defining event in the pathogenesis of Alzheimer's disease. To gain insight into disease pathogenesis, the mechanisms that trigger and mediate tau aggregation are under intense investigation. Characterization efforts have relied primarily on recombinant tau protein preparations and high-throughput solution-based detection methods such as thioflavin-dye fluorescence and laser-light-scattering spectroscopies. Transmission electron microscopy (TEM) is a static imaging tool that complements these approaches by detecting individual tau filaments at nanometer resolution. In doing so, it can provide unique insight into the quality, quantity, and composition of synthetic tau filament populations. Here we describe protocols for analysis of tau filament populations by TEM for purposes of dissecting aggregation mechanism.

Structure and mechanism of action of tau aggregation inhibitors.

Since the discovery of phenothiazines as tau protein aggregation inhibitors, many additional small molecule inhibitors of diverse chemotype have been discovered and characterized in biological model systems. Although direct inhibition of tau aggregation has shown promise as a potential treatment strategy for depressing neurofibrillary lesion formation in Alzheimer's disease, the mechanism of action of these compounds has been unclear. However, recent studies have found that tau aggregation antagonists exert their effects through both covalent and non-covalent means, and have identified associated potency and selectivity driving features. Here we review small-molecule tau aggregation inhibitors with a focus on compound structure and inhibitory mechanism. The elucidation of inhibitory mechanism has implications for maximizing on-target efficacy while minimizing off-target side effects.

Structural determinants of Tau aggregation inhibitor potency.

Small-molecule Tau aggregation inhibitors are under investigation as potential therapeutic agents against Alzheimer disease. Many such inhibitors have been identified in vitro, but their potency-driving features, and their molecular targets in the Tau aggregation pathway, have resisted identification. Previously we proposed ligand polarizability, a measure of electron delocalization, as a candidate descriptor of inhibitor potency. Here we tested this hypothesis by correlating the ground state polarizabilities of cyanine, phenothiazine, and arylmethine derivatives calculated using ab initio quantum methods with inhibitory potency values determined in the presence of octadecyl sulfate inducer under reducing conditions. A series of rhodanine analogs was analyzed as well using potency values disclosed in the literature. Results showed that polarizability and inhibitory potency directly correlated within all four series. To identify putative binding targets, representative members of the four chemotypes were added to aggregation reactions, where they were found to stabilize soluble, but SDS-resistant Tau species at the expense of filamentous aggregates. Using SDS resistance as a secondary assay, and a library of Tau deletion and missense mutants as targets, interaction with cyanine was localized to the microtubule binding repeat region. Moreover, the SDS-resistant phenotype was completely dependent on the presence of octadecyl sulfate inducer, but not intact PHF6/PH6* hexapeptide motifs, indicating that cyanine interacted with a species in the aggregation pathway prior to nucleus formation. Together the data suggest that flat, highly polarizable ligands inhibit Tau aggregation by interacting with folded species in the aggregation pathway and driving their assembly into soluble but highly stable Tau oligomers.