Tau Aggregation Propensity Engrained in Its Solution State.

A peptide fragment of the human tau protein which stacks to form neat cross β-sheet fibrils, resembling that found in pathological aggregation, (273)GKVQIINKKLDL(284) (here "R2/WT"), was modified with a spin-label at the N-terminus. With the resulting peptide, R2/G273C-SL, we probed events at time scales spanning seconds to hours after aggregation is initiated using transmission electron microscopy (TEM), thioflavin T (THT) fluorescence, ion mobility mass spectrometry (IMMS), electron paramagnetic resonance (EPR), and Overhauser dynamic nuclear polarization (ODNP) to determine if deliberate changes to its conformational states and population in solution influence downstream propensity to form fibrillar aggregates. We find varying solution conditions by adding the osmolyte urea or TMAO, or simply using different buffers (acetate buffer, phosphate buffer, or water), produces significant differences in early monomer/dimer populations and conformations. Crucially, these characteristics of the peptide in solution state before aggregation is initiated dictate the fibril formation propensity after aggregation. We conclude the driving forces that accelerate aggregation, when heparin is added, do not override the subtle intra- or interprotein interactions induced by the initial solvent conditions. In other words, the balance of protein-protein vs protein-solvent interactions present in the initial solution conditions is a critical driving force for fibril formation

RNA stores tau reversibly in complex coacervates.

Nonmembrane-bound organelles that behave like liquid droplets are widespread among eukaryotic cells. Their dysregulation appears to be a critical step in several neurodegenerative conditions. Here, we report that tau protein, the primary constituent of Alzheimer neurofibrillary tangles, can form liquid droplets and therefore has the necessary biophysical properties to undergo liquid-liquid phase separation (LLPS) in cells. Consonant with the factors that induce LLPS, tau is an intrinsically disordered protein that complexes with RNA to form droplets. Uniquely, the pool of RNAs to which tau binds in living cells are tRNAs. This phase state of tau is held in an approximately 1:1 charge balance across the protein and the nucleic acid constituents, and can thus be maximal at different RNA:tau mass ratios, depending on the biopolymer constituents involved. This feature is characteristic of complex coacervation. We furthermore show that the LLPS process is directly and sensitively tuned by salt concentration and temperature, implying it is modulated by both electrostatic interactions between the involved protein and nucleic acid constituents, as well as net changes in entropy. Despite the high protein concentration within the complex coacervate phase, tau is locally freely tumbling and capable of diffusing through the droplet interior. In fact, tau in the condensed phase state does not reveal any immediate changes in local protein packing, local conformations and local protein dynamics from that of tau in the dilute solution state. In contrast, the population of aggregation-prone tau as induced by the complexation with heparin is accompanied by large changes in local tau conformations and irreversible aggregation. However, prolonged residency within the droplet state eventually results in the emergence of detectable β-sheet structures according to thioflavin-T assay. These findings suggest that the droplet state can incubate tau and predispose the protein toward the formation of insoluble fibrils

RNA stores tau reversibly in complex coacervates.

Nonmembrane-bound organelles that behave like liquid droplets are widespread among eukaryotic cells. Their dysregulation appears to be a critical step in several neurodegenerative conditions. Here, we report that tau protein, the primary constituent of Alzheimer neurofibrillary tangles, can form liquid droplets and therefore has the necessary biophysical properties to undergo liquid-liquid phase separation (LLPS) in cells. Consonant with the factors that induce LLPS, tau is an intrinsically disordered protein that complexes with RNA to form droplets. Uniquely, the pool of RNAs to which tau binds in living cells are tRNAs. This phase state of tau is held in an approximately 1:1 charge balance across the protein and the nucleic acid constituents, and can thus be maximal at different RNA:tau mass ratios, depending on the biopolymer constituents involved. This feature is characteristic of complex coacervation. We furthermore show that the LLPS process is directly and sensitively tuned by salt concentration and temperature, implying it is modulated by both electrostatic interactions between the involved protein and nucleic acid constituents, as well as net changes in entropy. Despite the high protein concentration within the complex coacervate phase, tau is locally freely tumbling and capable of diffusing through the droplet interior. In fact, tau in the condensed phase state does not reveal any immediate changes in local protein packing, local conformations and local protein dynamics from that of tau in the dilute solution state. In contrast, the population of aggregation-prone tau as induced by the complexation with heparin is accompanied by large changes in local tau conformations and irreversible aggregation. However, prolonged residency within the droplet state eventually results in the emergence of detectable β-sheet structures according to thioflavin-T assay. These findings suggest that the droplet state can incubate tau and predispose the protein toward the formation of insoluble fibrils

RNA Stores Tau Reversibly in Complex Coacervates

Nonmembrane-bound organelles that behave like liquid droplets are widespread among eukaryotic cells. Their dysregulation appears to be a critical step in several neurodegenerative conditions. Here, we report that tau protein, the primary constituent of Alzheimer neurofibrillary tangles, can form liquid droplets and therefore has the necessary biophysical properties to undergo liquid-liquid phase separation (LLPS) in cells. Consonant with the factors that induce LLPS, tau is an intrinsically disordered protein that complexes with RNA to form droplets. Uniquely, the pool of RNAs to which tau binds in living cells are tRNAs. This phase state of tau is held in an approximately 1:1 charge balance across the protein and the nucleic acid constituents, and can thus be maximal at different RNA:tau mass ratios, depending on the biopolymer constituents involved. This feature is characteristic of complex coacervation. We furthermore show that the LLPS process is directly and sensitively tuned by salt concentration and temperature, implying it is modulated by both electrostatic interactions between the involved protein and nucleic acid constituents, as well as net changes in entropy. Despite the high protein concentration within the complex coacervate phase, tau is locally freely tumbling and capable of diffusing through the droplet interior. In fact, tau in the condensed phase state does not reveal any immediate changes in local protein packing, local conformations and local protein dynamics from that of tau in the dilute solution state. In contrast, the population of aggregation-prone tau as induced by the complexation with heparin is accompanied by large changes in local tau conformations and irreversible aggregation. However, prolonged residency within the droplet state eventually results in the emergence of detectable β-sheet structures according to thioflavin-T assay. These findings suggest that the droplet state can incubate tau and predispose the protein toward the formation of insoluble fibrils

Signature of an aggregation-prone conformation of tau

The self-assembly of the microtubule associated tau protein into fibrillar cell inclusions is linked to a number of devastating neurodegenerative disorders collectively known as tauopathies. The mechanism by which tau self-assembles into pathological entities is a matter of much debate, largely due to the lack of direct experimental insights into the earliest stages of aggregation. We present pulsed double electron-electron resonance measurements of two key fibril-forming regions of tau, PHF6 and PHF6∗, in transient as aggregation happens. By monitoring the end-to-end distance distribution of these segments as a function of aggregation time, we show that the PHF6 (∗) regions dramatically extend to distances commensurate with extended β-strand structures within the earliest stages of aggregation, well before fibril formation. Combined with simulations, our experiments show that the extended β-strand conformational state of PHF6 (∗) is readily populated under aggregating conditions, constituting a defining signature of aggregation-prone tau, and as such, a possible target for therapeutic interventions.NIH (S10 RR028992); NIH Innovator award; NIH/NIGMS (R21EB022731-- P41-GM103521), NSF (MCB 1158577); NSF MRSEC Program (DMR 1121053); Center for Scientific Computing at the UCSB CNSI (CNS-0960316); Extreme Science and Engineering Discovery Environment-XSEDE - NSF (TG-MCA05S027--ACI-1053575); University of California; Santa Barbara; University of California, Office of the Presiden

Tau Aggregation Propensity Engrained in Its Solution State

A peptide fragment of the human tau protein which stacks to form neat cross β-sheet fibrils, resembling that found in pathological aggregation, ²⁷³GKVQ­IINK­KLDL²⁸⁴ (here “R2/WT”), was modified with a spin-label at the N-terminus. With the resulting peptide, R2/G273C-SL, we probed events at time scales spanning seconds to hours after aggregation is initiated using transmission electron microscopy (TEM), thioflavin T (THT) fluorescence, ion mobility mass spectrometry (IMMS), electron paramagnetic resonance (EPR), and Overhauser dynamic nuclear polarization (ODNP) to determine if deliberate changes to its conformational states and population in solution influence downstream propensity to form fibrillar aggregates. We find varying solution conditions by adding the osmolyte urea or TMAO, or simply using different buffers (acetate buffer, phosphate buffer, or water), produces significant differences in early monomer/dimer populations and conformations. Crucially, these characteristics of the peptide in solution state <i>before</i> aggregation is initiated dictate the fibril formation propensity <i>after</i> aggregation. We conclude the driving forces that accelerate aggregation, when heparin is added, do not override the subtle intra- or interprotein interactions induced by the initial solvent conditions. In other words, the balance of protein–protein vs protein–solvent interactions present in the initial solution conditions is a critical driving force for fibril formation

tRNA transfection accumulates sarkosyl insoluble tau in human-induced pluripotent stem cell (hiPSC) derived neurons.

<p>(A) Representative western blot of neurons harboring a P301L <i>tau</i> mutation transfected with tRNA, but not cells transfected in the absence of nucleic acids (Mock) present evidence for accumulation of sarkosyl insoluble tau (Ins.) as seen in the intense band in lane 9 labeled with PHF-1 antibody. Addition of tRNA to the lysis buffer is insufficient to increase the tau present in the insoluble fractions (compare Mock + tRNA, lane 6 to tRNA, lane 9). (B-C) Quantification of PHF-1 tau and β-actin level for both neurons harboring a P301L tau mutation (B) and neurons expressing wild type tau (C) are shown. Error bar represents standard error of the mean. * <i>p</i> < 0.05, *** <i>p</i> < 0.001, <i>n</i> > 3. The numerical data used in B and C are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002183#pbio.2002183.s010" target="_blank">S1 Data</a>.</p

Tau in condensed droplets assumes solution state properties.

<p>(A-C) Continuous wave electron paramagnetic resonance (cw EPR) spectra obtained at room temperature of 500 μM Δtau187-SL in droplets formed with 1.5 mg/ml poly(A) RNA (blue in A) and 1.5 mg/ml tRNA (green in B) is unaltered from solution before adding RNA (red in A and B). Cw EPR line shape upon adding 125 μM heparin (black in C) show dramatic line broadening (compared to red in C). Double electron-electron resonance (DEER) of Δtau187-SL₂ in droplets formed with 1.5 mg/ml poly(A) RNA (blue in D) and 1.5 mg/ml tRNA (green in E), as well as upon incubation with 137.5 μM heparin (black in F) compared to Δtau187-SL₂ in solution (red in D-F). The numerical data are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002183#pbio.2002183.s010" target="_blank">S1 Data</a>.</p

Tau and RNA forms droplet in vitro.

<p>(A) Bright field snapshots of droplets from 400 μM Δtau187 and 800 μg/ml poly(A) showing 2 droplets seamlessly fusing (highlighted with red circle). (B) Confocal microscopy image of Δtau187 labeled with Alexa-488 of the droplets from the same sample as in (A). 50 μM Alexa-488 labeled Δtau187 mixed with 350 μM MTSL labeled Δtau187 of droplets formed immediately after adding 800 μg/ml poly(A) RNA. Both (A) and (B) were sampled at room temperature and without added NaCl.</p