Fracture and Growth Are Competing Forces Determining the Fate of Conformers in Tau Fibril Populations.

Tau fibrils are pathological aggregates that can transfer between neurons and then recruit soluble Tau monomers by template-assisted conversion. The propagation of different fibril polymorphs is thought to be a contributing factor to phenotypic diversity in Alzheimer disease and other Tauopathies. We found that a homogeneous population of Tau fibrils composed of the truncated version K18 (residues 244-372) gradually converted to a new set of fibril conformers when subjected to multiple cycles of seeding and growth. Using double electron-electron resonance (DEER) spectroscopy, we observed that the distances between spin labels at positions 311 and 328 in the fibril core progressively decreased. The findings were corroborated by changes in turbidity, morphology, and protease sensitivity. Fibrils that were initially formed under stirring conditions exhibited an increased fragility compared with fibrils formed quiescently after multiple cycles of seeding. The quiescently formed fibrils were marked by accelerated growth. The difference in fragility and growth between the different conformers explains how the change in incubation condition could lead to the amplification of a minor subpopulation of fibrils. Under quiescent conditions where fibril breakage is minimal, faster growing fibrils have a selective advantage. The findings are of general importance as they suggest that changes in selective pressures during fibril propagation in the human brain could result in the emergence of new fibril conformers with varied clinicopathological consequences.

Room-temperature distance measurements of immobilized spin-labeled protein by DEER/PELDOR.

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer Tm between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the Tm for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label.

The energetics of a three-state protein folding system probed by high-pressure relaxation dispersion NMR spectroscopy.

The energetic and volumetric properties of a three-state protein folding system, comprising a metastable triple mutant of the Fyn SH3 domain, have been investigated using pressure-dependent (15) N-relaxation dispersion NMR from 1 to 2500 bar. Changes in partial molar volumes (ΔV) and isothermal compressibilities (ΔκT ) between all the states along the folding pathway have been determined to reasonable accuracy. The partial volume and isothermal compressibility of the folded state are 100 mL mol(-1) and 40 µL mol(-1) bar(-1) , respectively, higher than those of the unfolded ensemble. Of particular interest are the findings related to the energetic and volumetric properties of the on-pathway folding intermediate. While the latter is energetically close to the unfolded state, its volumetric properties are similar to those of the folded protein. The compressibility of the intermediate is larger than that of the folded state reflecting the less rigid nature of the former relative to the latter.

Amplification of Tau fibrils from minute quantities of seeds.

The propagation of Tau pathology in Alzheimer's disease (AD) is thought to proceed through templated conversion of Tau protein into fibrils and cell-to-cell transfer of elongation-competent seeds. To investigate the efficiency of Tau conversion, we adapted the protein misfolding cyclic amplification assay used for the conversion of prions. Utilizing heparin as a cofactor and employing repetitive cycles of shearing and growth, synthetic Tau fibrils and Tau fibrils in AD brain extract are progressively amplified. Concurrently, self-nucleation is suppressed. The results highlight breakage-induced replication of Tau fibrils as a potential facilitator of disease spread.

Single mutations in tau modulate the populations of fibril conformers through seed selection.

Seeded conversion of tau monomers into fibrils is a central step in the progression of tau pathology in Alzheimer's disease and other neurodegenerative disorders. Self-assembly is mediated by the microtubule binding repeats in tau. There are either three or four repeats present depending on the protein isoform. Here, double electron-electron resonance spectroscopy was used to investigate the conformational ensemble of four-repeat tau fibrils. Single point mutations at key positions in the protein (ΔK280, P301S, P312I, D314I) markedly change the distribution of fibril conformers after template-assisted growth, whereas other mutations in the protein (I308M, S320F, G323I, G326I, Q336R) do not. These findings provide unprecedented insights into the seed selection of tau disease mutants and establish conformational compatibility as an important driving force in tau fibril propagation.

Temperature Dependence of Electron Spin Relaxation of 2,2-diphenyl-1-picrylhydrazyl in Polystyrene.

The electron spin relaxation rates for the stable radical DPPH (2,2-diphenyl-1-picrylhydrazyl) doped into polystyrene were studied by inversion recovery and electron spin echo at X-band and Q-band between 20 and 295 K. At low concentration (340 µM, 0.01%) spin-lattice relaxation was dominated by the Raman process and a local mode. At high concentration (140 mM, 5%) relaxation is orders of magnitude faster than at the lower concentration, and 1/T1 is approximately linearly dependent on temperature. Spin lattice relaxation rates are similar at X-band and Q-band. The temperature dependence of spin echo dephasing was faster at about 140 K than at higher or lower temperatures, which is attributed to a wagging motion of the phenyl groups.

Conformational basis for asymmetric seeding barrier in filaments of three- and four-repeat tau.

Tau pathology in Alzheimer's disease is intimately linked to the deposition of proteinacious filaments, which akin to infectious prions, have been proposed to spread via seeded conversion. Here we use double electron-electron resonance (DEER) spectroscopy in combination with extensive computational analysis to show that filaments of three- (3R) and four-repeat (4R) tau are conformationally distinct. Distance measurements between spin labels in the third repeat, reveal tau amyloid filaments as ensembles of known ß-strand-turn-ß-strand U-turn motifs. Whereas filaments seeded with 3R tau are structurally homogeneous, filaments seeded with 4R tau are heterogeneous, composed of at least three distinct conformers. These findings establish a molecular basis for the seeding barrier between different tau isoforms and offer a new powerful approach for investigating the composition and dynamics of amyloid fibril ensembles.

Optimizing COPD Acute Care Patient Outcomes Using a Standardized Transition Bundle and Care Coordinator: A Randomized Clinical Trial.

BACKGROUND: Acute exacerbations of COPD (AECOPD) are associated with high morbidity and mortality and frequent readmissions. RESEARCH QUESTION: What is the effectiveness of a COPD transition bundle, with and without a care coordinator, on rehospitalizations and ED revisits? STUDY DESIGN AND METHODS: Two patient cohorts were selected: (1) the group exposed to the transition bundle and (2) the group not exposed to the transition bundle (usual care group). Patients exposed subsequently were randomized to a care coordinator. An AECOPD transition bundle was implemented in the hospital; patients randomized to the care coordinator were contacted ≤ 72 h after discharge. Six hundred four patients (320 to the care coordinator and 284 to routine care) who met eligibility criteria from five hospitals across three cities in Alberta, Canada, were exposed to the transition bundle, whereas 3,106 patients discharged from the same hospitals received the usual care. Primary outcomes were 7-day, 30-day, and 90-day readmissions, median length of stay (LOS), and 30-day ED revisits. RESULTS: The transition bundle cohort were 83% (relative risk [RR], 0.17; 95% CI, 0.07-0.35) less likely to be readmitted within 7 days and 26% (RR, 0.74; 95% CI, 0.60-0.91) less likely to be readmitted within 30 days of discharge. Ninety-day readmissions were unchanged (RR, 1.05; 95% CI, 0.93-1.18). The transition bundle was associated with a 7.3% (RR, 1.07; 95% CI, 1.0-1.15) relative increase in LOS and a 76% (RR, 1.76; 95% CI, 1.53-2.02) greater risk of a 30-day ED revisit. The care coordinator did not influence readmission or ED revisits. INTERPRETATION: The COPD transition bundle reduced 7- and 30-day hospital readmissions while increasing LOS and ED revisits. The care coordinator did not improve outcomes. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03358771; URL: www. CLINICALTRIALS: gov.

Comparison of Continuous Wave, Spin Echo, and Rapid Scan EPR of Irradiated Fused Quartz.

The E' defect in irradiated fused quartz has spin lattice relaxation times (T(1)) about 100 to 300 µs and spin-spin relaxation times (T(2)) up to about 200 µs, depending on the concentration of defects and other species in the sample. These long relaxation times make it difficult to record an unsaturated continuous wave (CW) electron paramagnetic resonance (EPR) signal that is free of passage effects. Signals measured at X-band (~9.5 GHz) by three EPR methods: conventional slow-scan field modulated EPR, rapid scan EPR, and pulsed EPR, were compared. To acquire spectra with comparable signal-to-noise, both pulsed and rapid scan EPR require less time than conventional CW EPR. Rapid scan spectroscopy does not require the high power amplifiers that are needed for pulsed EPR. The pulsed spectra, and rapid scan spectra obtained by deconvolution of the experimental data, are free of passage effects.

Spin Labeling and Characterization of Tau Fibrils Using Electron Paramagnetic Resonance (EPR).

Template-assisted propagation of Tau fibrils is essential for the spreading of Tau pathology in Alzheimer's disease. In this process, small seeds of fibrils recruit Tau monomers onto their ends. The physical properties of the fibrils play an important role in their propagation. Here, we describe two different electron paramagnetic resonance (EPR) techniques that have provided crucial insights into the structure of Tau fibrils. Both techniques rely on the site-directed introduction of one or two spin labels into the protein monomer. Continuous-wave (CW) EPR provides information on which amino acid residues are contained in the fibril core and how they are stacked along the long fibril axis. Double electron-electron resonance (DEER) determines distances between two spin labels within a single protein and hence provides insights into their spatial arrangement in the fibril cross section. Because of the long distance range accessible to DEER (~2-5 nm) populations of distinct fibril conformers can be differentiated.

Electron spin-lattice relaxation mechanisms of rapidly-tumbling nitroxide radicals.

Electron spin relaxation times at 295 K were measured at frequencies between 250 MHz and 34 GHz for perdeuterated 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (PDT) in five solvents with viscosities that result in tumbling correlation times, τR, between 4 and 50 ps and for three (14)N/(15)N pairs of nitroxides in water with τR between 9 and 19 ps. To test the impact of structure on relaxation three additional nitroxides with τR between 10 and 26 ps were studied. In this fast tumbling regime T2(-1)~T1(-1) at frequencies up to about 9 GHz. At 34 GHz T2(-1)>T1(-1) due to increased contributions to T2(-1) from incomplete motional averaging of g-anisotropy, and T2(-1)-T1(-1) is proportional to τR. The contribution to T1(-1) from spin rotation is independent of frequency and decreases as τR increases. Spin rotation dominates T1(-1) at 34 GHz for all τR studied, and at all frequencies studied for τR=4 ps. The contribution to T1(-1) from modulation of nitrogen hyperfine anisotropy increases as frequency decreases and as τR increases; it dominates at low frequencies for τR>~15 ps. The contribution from modulation of g anisotropy is significant only at 34 GHz. Inclusion of a thermally-activated process was required to account for the observation that for most of the radicals, T1(-1) was smaller at 250 MHz than at 1-2 GHz. The significant (15)N/(14)N isotope effect, the small H/D isotope effect, and the viscosity dependence of the magnitude of the contribution from the thermally-activated process suggest that it arises from intramolecular motions of the nitroxide ring that modulate the isotropic A values.

Frequency dependence of electron spin relaxation times in aqueous solution for a nitronyl nitroxide radical and perdeuterated-tempone between 250 MHz and 34 GHz.

Electron spin relaxation times of perdeuterated tempone (PDT) 1 and of a nitronyl nitroxide (2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl) 2 in aqueous solution at room temperature were measured by 2-pulse electron spin echo (T(2)) or 3-pulse inversion recovery (T(1)) in the frequency range of 250 MHz to 34 GHz. At 9 GHz values of T(1) measured by long-pulse saturation recovery were in good agreement with values determined by inversion recovery. Below 9 GHz for 1 and below 1.5 GHz for 2,T(1)~T(2), as expected in the fast tumbling regime. At higher frequencies T(2) was shorter than T(1) due to incomplete motional averaging of g and A anisotropy. The frequency dependence of 1/T(1) is modeled as the sum of spin rotation, modulation of g and A-anisotropy, and a thermally-activated process that has maximum contribution at about 1.5 GHz. The spin lattice relaxation times for the nitronyl nitroxide were longer than for PDT by a factor of about 2 at 34 GHz, decreasing to about a factor of 1.5 at 250 MHz. The rotational correlation times, τ(R) are calculated to be 9 ps for 1 and about 25 ps for 2. The longer spin lattice relaxation times for 2 than for 1 at 9 and 34 GHz are due predominantly to smaller contributions from spin rotation that arise from slower tumbling. The smaller nitrogen hyperfine couplings for the nitronyl 2 than for 1 decrease the contribution to relaxation due to modulation of A anisotropy. However, at lower frequencies the slower tumbling of 2 results in a larger value of ωτ(R) (ω is the resonance frequency) and larger values of the spectral density function, which enhances the contribution from modulation of anisotropic interactions for 2 to a greater extent than for 1.

Relaxation times and line widths of isotopically-substituted nitroxides in aqueous solution at X-band.

Optimization of nitroxides as probes for EPR imaging requires detailed understanding of spectral properties. Spin lattice relaxation times, spin packet line widths, nuclear hyperfine splitting, and overall lineshapes were characterized for six low molecular weight nitroxides in dilute deoxygenated aqueous solution at X-band. The nitroxides included 6-member, unsaturated 5-member, or saturated 5-member rings, most of which were isotopically labeled. The spectra are near the fast tumbling limit with T(1)∼T(2) in the range of 0.50-1.1 µs at ambient temperature. Both spin-lattice relaxation T(1) and spin-spin relaxation T(2) are longer for (15)N- than for (14)N-nitroxides. The dominant contributions to T(1) are modulation of nitrogen hyperfine anisotropy and spin rotation. Dependence of T(1) on nitrogen nuclear spin state m(I) was observed for both (14)N and (15)N. Unresolved hydrogen/deuterium hyperfine couplings dominate overall line widths. Lineshapes were simulated by including all nuclear hyperfine couplings and spin packet line widths that agreed with values obtained by electron spin echo. Line widths and relaxation times are predicted to be about the same at 250 MHz as at X-band.

Electron spin relaxation and heterogeneity of the 1:1 α,γ-bisdiphenylene-ß-phenylallyl (BDPA)/benzene complex.

The electron spin-spin relaxation time (T(2)) for the 1:1 crystalline complex of α,γ-bisdiphenylene-ß-phenylallyl (BDPA) with benzene was determined by continuous wave (CW) and rapid scan electron paramagnetic resonance (EPR). T(2) for individual BDPA particles found by simulation of rapid scan spectra or by simulation of the Lorentzian line shapes of CW spectra were in good agreement. The T(2) for small BDPA particles in air ranged from 80 to 160 ns, which corresponds to peak-to-peak Lorentzian linewidths of 0.82-0.41 G. The removal of oxygen from the samples had a greater impact on the line width for particles that had shorter T(2) in air. Heterogeneity in the g-value was not observed at X-band. Scanning electron microscope (SEM) images showed that the BDPA particles had varying morphology.