Edward Chang

Direct charging of tRNA(CUA) with pyrrolysine in vitro and in vivo.

Pyrrolysine is the 22nd amino acid. An unresolved question has been how this atypical genetically encoded residue is inserted into proteins, because all previously described naturally occurring aminoacyl-tRNA synthetases are specific for one of the 20 universally distributed amino acids. Here we establish that synthetic l-pyrrolysine is attached as a free molecule to tRNACUA by PylS, an archaeal class II aminoacyl-tRNA synthetase. PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNACUA in vitro in reactions specific for pyrrolysine. The addition of pyrrolysine to Escherichia coli cells expressing pylT (encoding tRNACUA) and pylS results in the translation of UAG in vivo as a sense codon. This is the first example from nature of direct aminoacylation of a tRNA with a non-canonical amino acid and shows that the genetic code of E. coli can be expanded to include UAG-directed pyrrolysine incorporation into proteins.

Pathogenic missense MAPT mutations differentially modulate tau aggregation propensity at nucleation and extension steps

Mutations in the MAPT gene encoding tau protein lead to neurofibrillary lesion formation, neurodegeneration, and cognitive decline associated with frontotemporal lobar degeneration. While some pathogenic mutations affect MAPT introns, resulting in abnormal splicing patterns, the majority occur in the tau coding sequence leading to single amino acid changes in tau primary structure. Depending on their location within the polypeptide chain, tau missense mutations have been reported to augment aggregation propensity. To determine the mechanisms underlying mutation‐associated changes in aggregation behavior, the fibrillization of recombinant pathogenic mutants R5L, G272V, P301L, V337M, and R406W prepared in a full‐length four‐repeat human 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. Results indicated that the mutants differ from each other and from wild‐type tau in their aggregation propensity. G272V and P301L mutations increased the rates of both filament nucleation and extension reactions, whereas R5L and V337M increased only the nucleation phase. R406W did not differ from wild‐type in any kinetic parameter. The results show that missense mutations can directly promote tau filament formation at different stages of the aggregation pathway.

Inhibition of tau polymerization with a cyanine dye in two distinct model systems

In a host of neurodegenerative diseases Tau, a microtubule-associated protein, aggregates into insoluble lesions within neurons. Previous studies have utilized cyanine dyes as Tau aggregation inhibitors in vitro. Herein we utilize cyanine dye 3,3′-diethyl-9-methyl-thiacarbocyanine iodide (C11) to modulate Tau polymerization in two model systems, an organotypic slice culture model derived from Tau transgenic mice and a split green fluorescent protein complementation assay in Tau-expressing cells. In slice cultures, submicromolar concentrations (0.001 μm) of C11 produced a significant reduction of aggregated Tau and a corresponding increase in unpolymerized Tau. In contrast, treatment with a 1 μm dose promoted aggregation of Tau. These results were recapitulated in the complementation assay where administration of 1 μm C11 produced a significant increase in polymerized Tau relative to control, whereas treatment of cells with 0.01 μm C11 resulted in a marked reduction of aggregated Tau. In the organotypic slice cultures, modulation of Tau aggregation was independent of changes in phosphorylation at disease and microtubule binding relevant epitopes for both dosing regimes. Furthermore, treatment with 0.001 μm C11 resulted in a decrease in both total filament mass and number. There was no evidence of apoptosis or loss of synaptic integrity at either dose, however, whereas submicromolar concentrations of C11 did not interfere with microtubule binding, higher doses resulted in a decrease in the levels of microtubule-bound Tau. Overall, a cyanine dye can dissociate aggregated Tau in an ex vivo model of tauopathy with little toxicity and exploration of the use of these type of dyes as therapeutic agents is warranted.

Structural Determinants of Tau Aggregation Inhibitor Potency

Background: Mechanistic insight into small-molecule Tau aggregation inhibitors is needed for their advancement as therapeutic agents. Results: Structure-activity relationship analysis identified polarizability as a common descriptor of inhibitor potency. Conclusion: Flat, highly polarizable ligands stabilize soluble oligomeric complexes at the expense of filamentous aggregates. Significance: The findings suggest a basis for rational improvement of ligand potency, whereas maintaining bioavailability. 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.

Pseudophosphorylation of tau protein directly modulates its aggregation kinetics

Hyperphosphorylation of tau protein is associated with neurofibrillary lesion formation in Alzheimers 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.

Modulation and detection of tau aggregation with small-molecule ligands.

Recent results from high-throughput and other screening approaches reveal that small molecules can directly interact with recombinant full-length tau monomers and fibrillar tau aggregates in three distinct modes. First, in the high concentration regime (>10 micromolar), certain anionic molecules such as Congo red efficiently promote tau filament formation through a nucleation-elongation mechanism involving a dimeric nucleus and monomer-mediated elongation. These compounds are useful for modeling tau aggregation in vitro and in biological models. Second, in the low concentration regime (<1 micromolar), other ligands, including cyanine dyes, display aggregation antagonist activity. Compounds that can prevent or reverse fibrillization are candidate modifiers of disease pathology. Finally, certain compounds bind mature tau fibrils with varying affinities at multiple binding sites without modulating the aggregation reaction. For some ligands, >10-fold selectivity for tau aggregates relative to filaments composed of beta-amyloid or alpha-synuclein can be demonstrated at the level of binding affinity. Together these observations suggest that small-molecules have utility for interrogating the tau aggregation pathway, for inhibiting neuritic lesion formation, and for selective pre-mortem detection of neurofibrillary lesions through whole brain imaging.

The metabolism in vitro of [4-14C]cortisone by lymphosarcoma P1798

The metabolism in vitro of [4-14C]cortisone by homogenates of cortisone-sensitive and resistant lines of lymphosarcoma P1798 was studied. The administration of cortisol for several days to mice bearing these tumors significantly altered the capacity of both tumor lines to metabolize cortisone. Following such treatment, the cortisone-sensitive tumor appears to be more efficient in converting cortisone to cortisol, while the resistant neoplasm is better able to convert cortisone to 20β-hydroxycortisone (Reuchsteins substance “U”) which is biologically inactive. The results suggest that the enzymes which mediate the formation of these metabolites may be responsive to glucocorticoid administration.

P2-179: Small-molecule probes of tau fibrillization

memory formation--in PrP-MAPT mice that over-express normal human fetal tau (3R/0N isoform). Methods: Field potential recordings were made from the lateral perforant path of the dentate gyrus of either wild-type or heterozygous mice aged 2 and 4 months, respectively, and include assessments of basal transmission, paired-pulse facilitation (PPF) and long-term potentiation (LTP). Results: At 2 months, no difference in the magnitude of LTP between genotypes was observed (Het: 176.4 10.2%, n 6; WT: 171.3 9.1, n 6) but a subtle impairment in basal transmission was detected in the Het’s even at this early age. By 4 months of age, differences in basal transmission, PPF, and LTP (WT: 178.2 6.4, n 6; Het: 148.1 11.9, n 9) were observed. Interestingly, 4M Het LTP data revealed that some slices had normal, WT LTP magnitudes while others were impaired. This duality suggests that at 4 months, Het mice may reside on a threshold between normal and abnormal and that other factors influence the progression of symptoms. Further studies will include assessments in 8-10M old Tau transgenic mice. Conclusions: These data suggest that overexpression of normal tau protein influences hippocampal synaptic properties, similar to deficits found in Tg2576 mice, early in development and thus may be important in the disease progression.