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.

Selectivity requirements for diagnostic imaging of neurofibrillary lesions in Alzheimer's disease: a simulation study.

Whole-brain imaging is a promising strategy for premortem detection of tau-bearing neurofibrillary lesions that accumulate in Alzheimer's disease. However, the approach is complicated by the high concentrations of potentially confounding binding sites presented by beta-amyloid plaques. To predict the contributions of relative binding affinity and binding site density to the imaging-dynamics and selectivity of a hypothetical tau-directed radiotracer, a nonlinear, four-tissue compartment pharmacokinetic model of diffusion-mediated radiotracer uptake and distribution was developed. Initial estimates of nonspecific binding and brain uptake parameters were made by fitting data from a previously published kinetic study of Pittsburgh Compound B, an established amyloid-directed radiotracer. The resulting estimates were then used to guide simulations of tau binding selectivity while assuming early-stage accumulation of disease pathology. The simulations suggest that for tau aggregates to represent at least 80% of specific binding signal, binding affinity or density selectivities for tau over beta-amyloid should be at least 20- or 50-fold, respectively. The simulations also suggest, however, that overcoming nonspecific binding will be an additional challenge for tau-directed radiotracers owing to low concentrations of available binding sites. Overall, nonlinear modeling can provide insight into the performance characteristics needed for tau-directed radiotracers in vivo.

Research towards tau imaging.

Tau-bearing neurofibrillary lesions present a promising biomarker for premortem diagnosis and staging of Alzheimer's disease and certain forms of frontotemporal lobar degeneration by whole brain imaging methods. Although brain penetrating compounds capable of binding tau aggregates with high affinity have been disclosed for this purpose, the major barrier to progress remains the need for tau lesion binding selectivity relative to amyloid-beta plaques and other deposits of proteins in cross-beta-sheet conformation. Here we discuss challenges faced in the development of tau lesion-selective imaging agents, and recent preclinical advances in pursuit of this goal.

Structure-activity relationship of cyclic thiacarbocyanine tau aggregation inhibitors.

Macrocyclic bis-thiacarbocyanines are efficacious inhibitors of tau protein aggregation. To extend the structure-activity relationship of this inhibitor class, N,N'-alkylene bis-thiacarbocyanines linked by chains of three to eight methylene carbons were synthesized and examined for inhibitory activity against recombinant human tau aggregation in vitro. At 10 micromolar concentration, inhibitory activity varied with linker length, with four methylene units being most efficacious. On the basis of absorbance spectroscopy measurements, linker length also affected compound folding and aggregation propensity, with a linker length of four methylene units being optimal for preserving open monomer conformation. These data suggest that inhibitory potency can be optimized through control of linker length, and that a contributory mechanism involves modulation of compound folding and aggregation.

Pseudophosphorylation of tau protein directly modulates its aggregation kinetics.

Hyperphosphorylation of tau protein is associated with neurofibrillary lesion formation in Alzheimer's disease and other tauopathic neurodegenerative diseases. It fosters lesion formation by increasing the concentration of free tau available for aggregation and by directly modulating the tau aggregation reaction. To clarify how negative charge incorporation into tau directly affects aggregation behavior, the fibrillization of pseudophosphorylation mutant T212E prepared in a full-length four-repeat tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Kinetic analysis revealed that pseudophosphorylation increased tau aggregation rate by increasing the rate of filament nucleation. In addition, it increased aggregation propensity by stabilizing mature filaments against disaggregation. The data suggest that incorporation of negative charge into the T212 site can directly promote tau filament formation at multiple steps in the aggregation pathway.