Quantification of Methylation and Phosphorylation Stoichiometry.

Tauopathies including Alzheimer's disease (AD) are neurodegenerative disorders accompanied by the conversion of functional forms of the microtubule associated protein Tau into non-functional aggregates. A variety of post-translational modifications (PTMs) on Tau precede or accompany the conversion, placing them in position to modulate Tau function as well as its propensity to aggregate. Although Tau PTMs can be characterized by their sites of modification, their total stoichiometry when summed over all sites also is an important metric of their potential impact on function. Here we provide a protocol for rapidly producing recombinant Tau with enzyme-specific PTMs at high stoichiometry in vitro and demonstrate its utility in the context of hyperphosphorylation. Additionally, protocols for estimating phosphorylation and methylation stoichiometry on Tau proteins isolated from any source are presented. Together these methods support experimentation on Tau PTM function over a wide range of experimental conditions.

Sedimentation and Laser Light Scattering Methods for Quantifying Synthetic Tau Aggregation Propensity.

Tau aggregation assays detect and quantify the conversion of soluble tau monomers into species having filamentous or oligomeric structure. Assays for filamentous aggregates in cross-ß-sheet conformation leverage optical, biochemical, or biophysical methods, each with their own advantages and throughput capacity. Here we provide protocols for two medium-throughput assays based on sedimentation and laser light scattering and compare their performance, their utility for characterizing tau aggregation dynamics, and their limitations relative to other approaches. Additionally, a protocol for transmission electron microscopy analysis is updated so as to be compatible with the truncated tau variants that have emerged as powerful tools for interrogating the structural basis of tau polymorphism. Together these methods contribute to a rich tool kit for interrogating tau aggregation kinetics and propensity over a wide range of experimental conditions.

Identification of gene networks mediating regional resistance to tauopathy in late-onset Alzheimer's disease.

Neurofibrillary lesions composed of tau protein aggregates are defining hallmarks of Alzheimer's Disease. Despite tau filaments appearing to spread between networked brain regions in a prion-like manner, certain areas including cerebellum resist trans-synaptic spread of tauopathy and degeneration of their constituent neuronal cell bodies. To identify molecular correlates of resistance, we derived and implemented a ratio of ratios approach for disaggregating gene expression data on the basis of regional vulnerability to tauopathic neurodegeneration. When applied to vulnerable pre-frontal cortex as an internal reference for resistant cerebellum, the approach segregated adaptive changes in expression into two components. The first was enriched for neuron-derived transcripts associated with proteostasis including specific members of the molecular chaperone family and was unique to resistant cerebellum. When produced as purified proteins, each of the identified chaperones depressed aggregation of 2N4R tau in vitro at sub-stoichiometric concentrations, consistent with the expression polarity deduced from ratio of ratios testing. In contrast, the second component enriched for glia- and microglia-derived transcripts associated with neuroinflammation, segregating these pathways from susceptibility to tauopathy. These data support the utility of ratio of ratios testing for establishing the polarity of gene expression changes with respect to selective vulnerability. The approach has the potential to identify new targets for drug discovery predicated on their ability to promote resistance to disease in vulnerable neuron populations.

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.

Wolframin is a novel regulator of tau pathology and neurodegeneration.

Selective neuronal vulnerability to protein aggregation is found in many neurodegenerative diseases including Alzheimer's disease (AD). Understanding the molecular origins of this selective vulnerability is, therefore, of fundamental importance. Tau protein aggregates have been found in Wolframin (WFS1)-expressing excitatory neurons in the entorhinal cortex, one of the earliest affected regions in AD. The role of WFS1 in Tauopathies and its levels in tau pathology-associated neurodegeneration, however, is largely unknown. Here we report that WFS1 deficiency is associated with increased tau pathology and neurodegeneration, whereas overexpression of WFS1 reduces those changes. We also find that WFS1 interacts with tau protein and controls the susceptibility to tau pathology. Furthermore, chronic ER stress and autophagy-lysosome pathway (ALP)-associated genes are enriched in WFS1-high excitatory neurons in human AD at early Braak stages. The protein levels of ER stress and autophagy-lysosome pathway (ALP)-associated proteins are changed in tau transgenic mice with WFS1 deficiency, while overexpression of WFS1 reverses those changes. This work demonstrates a possible role for WFS1 in the regulation of tau pathology and neurodegeneration via chronic ER stress and the downstream ALP. Our findings provide insights into mechanisms that underpin selective neuronal vulnerability, and for developing new therapeutics to protect vulnerable neurons in AD.

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.

Hyperphosphorylation Renders Tau Prone to Aggregate and to Cause Cell Death.

Alzheimer's disease (AD) is a neurodegenerative disorder without a cure or prevention to date. Hyperphosphorylated tau forms the neurofibrillary tangles (NFTs) that correlate well with the progression of cognitive impairments. Animal studies demonstrated the pathogenic role of hyperphosphorylated tau. Understanding how abnormal phosphorylation renders a normal tau prone to form toxic fibrils is key to delineating molecular pathology and to developing efficacious drugs for AD. Production of a tau bearing the disease-relevant hyperphosphorylation and molecular characters is a pivotal step. Here, we report the preparation and characterization of a recombinant hyperphosphorylated tau (p-tau) with strong relevance to disease. P-tau generated by the PIMAX approach resulted in phosphorylation at multiple epitopes linked to the progression of AD neuropathology. In stark contrast to unmodified tau that required an aggregation inducer, and which had minimal effects on cell functions, p-tau formed inducer-free fibrils that triggered a spike of mitochondrial superoxide, induced apoptosis, and caused cell death at sub-micromolar concentrations. P-tau-induced apoptosis was suppressed by inhibitors for reactive oxygen species. Hyperphosphorylation apparently caused rapid formation of a disease-related conformation. In both aggregation and cytotoxicity, p-tau exhibited seeding activities that converted the unmodified tau into a cytotoxic species with an increased propensity for fibrillization. These characters of p-tau are consistent with the emerging view that hyperphosphorylation causes tau to become an aggregation-prone and cytotoxic species that underlies diffusible pathology in AD and other tauopathies. Our results further suggest that p-tau affords a feasible tool for Alzheimer's disease mechanistic and drug discovery studies.

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.

Membrane-specific spin trap, 5-dodecylcarbamoyl-5-N-dodecylacetamide-1-pyroline-N-oxide (diC12PO): theoretical, bioorthogonal fluorescence imaging and EPR studies.

Membranous organelles are major endogenous sources of reactive oxygen and nitrogen species. When present at high levels, these species can cause macromolecular damage and disease. To better detect and scavenge free radical forms of the reactive species at their sources, we investigated whether nitrone spin traps could be selectively targeted to intracellular membranes using a bioorthogonal imaging approach. Electron paramagnetic resonance imaging demonstrated that the novel cyclic nitrone 5-dodecylcarbamoyl-5-N-dodecylacetamide-1-pyroline-N-oxide (diC12PO) could be used to target the nitrone moiety to liposomes composed of phosphatidyl choline. To test localization with authentic membranes in living cells, fluorophores were introduced via strain-promoted alkyne-nitrone cycloaddition (SPANC). Two fluorophore-conjugated alkynes were investigated: hexynamide-fluoresceine (HYA-FL) and dibenzylcyclooctyne-PEG4-5/6-sulforhodamine B (DBCO-Rhod). Computational and mass spectrometry experiments confirmed the cycloadduct formation of DBCO-Rhod (but not HYA-FL) with diC12PO in cell-free solution. Confocal microscopy of bovine aortic endothelial cells treated sequentially with diC12PO and DBCO-Rhod demonstrated clear localization of fluorescence with intracellular membranes. These results indicate that targeting of nitrone spin traps to cellular membranes is feasible, and that a bioorthogonal approach can aid the interrogation of their intracellular compartmentalization properties.

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.

Computational integration of nanoscale physical biomarkers and cognitive assessments for Alzheimer's disease diagnosis and prognosis.

With the increasing prevalence of Alzheimer's disease (AD), significant efforts have been directed toward developing novel diagnostics and biomarkers that can enhance AD detection and management. AD affects the cognition, behavior, function, and physiology of patients through mechanisms that are still being elucidated. Current AD diagnosis is contingent on evaluating which symptoms and signs a patient does or does not display. Concerns have been raised that AD diagnosis may be affected by how those measurements are analyzed. Unbiased means of diagnosing AD using computational algorithms that integrate multidisciplinary inputs, ranging from nanoscale biomarkers to cognitive assessments, and integrating both biochemical and physical changes may provide solutions to these limitations due to lack of understanding for the dynamic progress of the disease coupled with multiple symptoms in multiscale. We show that nanoscale physical properties of protein aggregates from the cerebral spinal fluid and blood of patients are altered during AD pathogenesis and that these properties can be used as a new class of "physical biomarkers." Using a computational algorithm, developed to integrate these biomarkers and cognitive assessments, we demonstrate an approach to impartially diagnose AD and predict its progression. Real-time diagnostic updates of progression could be made on the basis of the changes in the physical biomarkers and the cognitive assessment scores of patients over time. Additionally, the Nyquist-Shannon sampling theorem was used to determine the minimum number of necessary patient checkups to effectively predict disease progression. This integrated computational approach can generate patient-specific, personalized signatures for AD diagnosis and prognosis.

Detection and Quantification Methods for Fibrillar Products of In Vitro Tau Aggregation Assays.

Alzheimer's disease is characterized in part by the intracellular misfolding and aggregation of tau protein. The aggregates, which range in size from small oligomers to large filaments, are markers for disease diagnosis and staging, potential vectors for disease propagation, and candidate sources of neurotoxicity. Here we present protocols for synthesizing large tau aggregates characterized by filamentous morphology and cross-ß-sheet structure from monomeric full-length tau precursors in vitro. We also describe their detection and quantification through thioflavin dye binding, filter trap, and transmission electron microscopy methods. These methods cover applications requiring high-throughput capability as well as those requiring high-resolution analysis of aggregation mechanism.

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.

Direct cellular delivery of human proteasomes to delay tau aggregation.

The 26S proteasome is the primary machinery that degrades ubiquitin (Ub)-conjugated proteins, including many proteotoxic proteins implicated in neurodegeneraton. It has been suggested that the elevation of proteasomal activity is tolerable to cells and may be beneficial to prevent the accumulation of protein aggregates. Here we show that purified proteasomes can be directly transported into cells through mesoporous silica nanoparticle-mediated endocytosis. Proteasomes that are loaded onto nanoparticles through non-covalent interactions between polyhistidine tags and nickel ions fully retain their proteolytic activity. Cells treated with exogenous proteasomes are more efficient in degrading overexpressed human tau than endogenous proteasomal substrates, resulting in decreased levels of tau aggregates. Moreover, exogenous proteasome delivery significantly promotes cell survival against proteotoxic stress caused by tau and reactive oxygen species. These data demonstrate that increasing cellular proteasome activity through the direct delivery of purified proteasomes may be an effective strategy for reducing cellular levels of proteotoxic proteins.

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.

Lysine methylation is an endogenous post-translational modification of tau protein in human brain and a modulator of aggregation propensity.

In Alzheimer's disease, the microtubule-associated protein tau dissociates from the neuronal cytoskeleton and aggregates to form cytoplasmic inclusions. Although hyperphosphorylation of tau serine and threonine residues is an established trigger of tau misfunction and aggregation, tau modifications extend to lysine residues as well, raising the possibility that different modification signatures depress or promote aggregation propensity depending on site occupancy. To identify lysine residue modifications associated with normal tau function, soluble tau proteins isolated from four cognitively normal human brains were characterized by MS methods. The major detectable lysine modification was found to be methylation, which appeared in the form of mono- and di-methyl lysine residues distributed among at least 11 sites. Unlike tau phosphorylation sites, the frequency of lysine methylation was highest in the microtubule-binding repeat region that mediates both microtubule binding and homotypic interactions. When purified recombinant human tau was modified in vitro through reductive methylation, its ability to promote tubulin polymerization was retained, whereas its aggregation propensity was greatly attenuated at both nucleation and extension steps. These data establish lysine methylation as part of the normal tau post-translational modification signature in human brain, and suggest that it can function in part to protect against pathological tau aggregation.

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.

Ligand electronic properties modulate tau filament binding site density.

Small molecules that bind tau-bearing neurofibrillary lesions are being sought for premortem diagnosis, staging, and treatment of Alzheimer's disease and other tauopathic neurodegenerative diseases. The utility of these agents will depend on both their binding affinity and binding site density (B(max)). Previously we identified polarizability as a descriptor of protein aggregate binding affinity. To examine its contribution to binding site density, we investigated the ability of two closely related benzothiazole derivatives ((E)-2-[[4-(dimethylamino)phenyl]azo]-6-methoxybenzothiazole) and ((E)-2-[2-[4-(dimethylamino)phenyl]ethenyl]-6-methoxybenzothiazole) that differed in polarizability to displace probes of high (Thioflavin S) and low (radiolabeled (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene; IMSB) density sites. Consistent with their site densities, Thioflavin S completely displaced radiolabeled IMSB, but IMSB was incapable of displacing Thioflavin S. Although both benzothiazoles displaced the low B(max) IMSB probe, only the highly polarizable analog displaced near saturating concentrations of the Thioflavin S probe. Quantum calculations showed that high polarizability reflected extensive pi-electron delocalization fostered by the presence of electron donating and accepting groups. These data suggest that electron delocalization promotes ligand binding at a subset of sites on tau aggregates that are present at high density, and that optimizing this aspect of ligand structure can yield tau-directed agents with superior diagnostic and therapeutic performance.

Lysosomal fusion dysfunction as a unifying hypothesis for Alzheimer's disease pathology.

Alzheimer's disease is characterized pathologically by extracellular senile plaques, intracellular neurofibrillary tangles, and granulovacuolar degeneration. It has been debated whether these hallmark lesions are markers or mediators of disease progression, and numerous paradigms have been proposed to explain the appearance of each lesion individually. However, the unfaltering predictability of these lesions suggests a single pathological nidus central to disease onset and progression. One of the earliest pathologies observed in Alzheimer's disease is endocytic dysfunction. Here we review the recent literature of endocytic dysfunction with particular focus on disrupted lysosomal fusion and propose it as a unifying hypothesis for the three most-studied lesions of Alzheimer's disease.