Modulation of aggregation and structural polymorphisms of ß-amyloid fibrils in cellular environments by pyroglutamate-3 variant cross-seeding.

Amyloidogenic deposition of ß-amyloid (Aß) peptides in human brain involves not only the wild-type Aß (wt-Aß) sequences, but also posttranslationally modified Aß (PTM-Aß) variants. Recent studies hypothesizes that the PTM-Aß variants may trigger the deposition of wt-Aß, which underlies the pathology of Sporadic Alzheimer's disease. Among PTM-Aß variants, the pyroglutamate-3-Aß (pyroE3-Aß) has attracted much attention because of their significant abundances and broad distributions in senile plaques and dispersible and soluble oligomers. pyroE3-specific antibodies are being tested as potential anti-Aß drugs in clinical trials. However, evidence that support the triggering effect of pyroE3-Aß on wt-Aß in cells remain lacking, which diminishes its pathological relevance. We show here that cross-seeding with pyroE3-Aß40 leads to accelerated extracellular and intracellular aggregation of wt-Aß40 in different neuronal cells. Cytotoxicity levels are elevated through the cross-seeded aggregation, comparing with the self-seeded aggregation of wt-Aß40 or the static presence of pyroE3-Aß40 seeds. For the extracellular deposition in mouse neuroblastoma Neuro2a (N2a) cells, the cytotoxicity elevation correlates positively with the seeding efficiency. Besides aggregation rates, cross-seeding with pyroE3-Aß40 also modulates the molecular level structural polymorphisms of the resultant wt-Aß40 fibrils. Using solid-state nuclear magnetic resonance (ssNMR) spectroscopy, we identified key structural differences between the parent pyroE3/ΔE3 and wt-Aß40 fibrils within their fibrillar cores. Structural propagation from seeds to daughter fibrils is demonstrated to be more pronounced in the extracellular seeding in N2a cells by comparing the ssNMR spectra from different seeded wt-Aß40 fibrils, but less significant in the intracellular seeding process in human neuroblastoma SH-SY5Y cells.

Persistence of Methionine Side Chain Mobility at Low Temperatures in a Nine-Residue Low Complexity Peptide, as Probed by 2 H Solid-State NMR.

Methionine side chains are flexible entities which play important roles in defining hydrophobic interfaces. We utilize deuterium static solid-state NMR to assess rotameric inter-conversions and other dynamic modes of the methionine in the context of a nine-residue random-coil peptide (RC9) with the low-complexity sequence GGKGMGFGL. The measurements in the temperature range of 313 to 161 K demonstrate that the rotameric interconversions in the hydrated solid powder state persist to temperatures below 200 K. Removal of solvation significantly reduces the rate of the rotameric motions. We employed 2 H NMR line shape analysis, longitudinal and rotation frame relaxation, and chemical exchange saturation transfer methods and found that the combination of multiple techniques creates a significantly more refined model in comparison with a single technique. Further, we compare the most essential features of the dynamics in RC9 to two different methionine-containing systems, characterized previously. Namely, the M35 of hydrated amyloid-ß1-40 in the three-fold symmetric polymorph as well as Fluorenylmethyloxycarbonyl (FMOC)-methionine amino acid with the bulky hydrophobic group. The comparison suggests that the driving force for the enhanced methionine side chain mobility in RC9 is the thermodynamic factor stemming from distributions of rotameric populations, rather than the increase in the rate constant.

Rigidifying of the internal dynamics of amyloid-beta fibrils generated in the presence of synaptic plasma vesicles.

We investigated the changes in internal flexibility of amyloid-ß1-40 (Aß) fibrils grown in the presence of rat synaptic plasma vesicles. The fibrils are produced using a modified seeded growth protocol, in which the Aß concentration is progressively increased at the expense of the decreased lipid to protein ratio. The morphologies of each generation are carefully assessed at several fibrils' growth time points using transmission electron microscopy. The side-chain dynamics in the fibrils is investigated using deuterium solid-state NMR measurements, with techniques spanning line shapes analysis and several NMR relaxation rates measurements. The dynamics is probed in the site-specific fashion in the hydrophobic C-terminal domain and the disordered N-terminal domain. An overall strong rigidifying effect is observed in comparison with the wild-type fibrils generated in the absence of the membranes. In particular, the overall large-scale fluctuations of the N-terminal domain are significantly reduced, and the activation energies of rotameric inter-conversion in methyl-bearing side-chains of the core (L17, L34, M35, V36), as well as the ring-flipping motions of F19 are increased, indicating a restricted core environment. Membrane-induced flexibility changes in Aß aggregates can be important for the re-alignment of protein aggregates within the membrane, which in turn would act as a disruption pathway of the bilayers' integrity.

Carbon-detected deuterium solid-state NMR rotating frame relaxation measurements for protein methyl groups under magic angle spinning.

Deuterium rotating frame solid-state NMR relaxation measurements (2H R1ρ) are important tools in quantitative studies of molecular dynamics. We demonstrate how 2H to 13C cross-polarization (CP) approaches under 10-40 kHz magic angle spinning rates can be combined with the 2H R1ρ blocks to allow for extension of deuterium rotating frame relaxation studies to methyl groups in biomolecules. This extension permits detection on the 13C nuclei and, hence, for the achievement of site-specific resolution. The measurements are demonstrated using a nine-residue low complexity peptide with the sequence GGKGMGFGL, in which a single selective -13CD3 label is placed at the methionine residue. Carbon-detected measurements are compared with the deuterium direct-detection results, which allows for fine-tuning of experimental approaches. In particular, we show how the adiabatic respiration CP scheme and the double adiabatic sweep on the 2H and 13C channels can be combined with the 2H R1ρ relaxation rates measurement. Off-resonance 2H R1ρ measurements are investigated in addition to the on-resonance condition, as they extent the range of effective spin-locking field.

Adult tonsillectomy-increased pain scores are correlated with risk of bleeding: a retrospective cohort study.

PURPOSE: Tonsillectomy is among the most common surgical procedures performed worldwide, and post-tonsillectomy bleeding is a serious complication. This study aims to investigate the role of post-operative pain as a risk factor for bleeding in adults. METHODS: A retrospective cohort study of adults who underwent tonsillectomy in a tertiary referral center between 2015-2021. Medical records were reviewed for demographics, diagnoses, surgical technique, treatments, pain scores (measured by visual analogue scale 0-10), readmissions, and bleeding events. The primary outcome was return to the operating room for hemostasis, and secondary outcomes were bleeding events and consumption of additional analgesic doses. RESULTS: Of the 274 patients, 137 (50%) were males, the mean age was 30.3 ± 12 years (range 18-82), and 33 (12%) were smokers. Indications for tonsillectomy were recurrent throat infections in 213 (77.7%) patients and obstructive sleep apnea in 61 (22.3%). Surgical technique was cold dissection in 238 (86.9%) patients and electrocautery in 36 (13.1%). Primary post-tonsillectomy bleeding (< 24 h of surgery) occurred in 6 (2%) patients, and secondary bleeding (later than 24 h from tonsillectomy) in 43 (15.7%). A total of 19 (7%) patients necessitated surgical hemostasis. After controlling for technique and other confounders, high pain scores (VAS ≥ 5) on post-operative days 1 and 2 were associated with increased risk of bleeding that necessitated surgical hemostasis (adjusted odds ratio 6.9, 95% confidence interval 1.7-44.5). Other independent risk factors were male sex, age < 30 years, smoking, and recurrent throat infections. CONCLUSIONS: Higher pain scores following tonsillectomy are correlated with bleeding episodes requiring surgical intervention in adults. Further studies may explore the role of different intensive pain regimens in minimizing the risk of bleeding.

Comparative Hydrophobic Core Dynamics Between Wild-Type Amyloid-ß Fibrils, Glutamate-3 Truncation, and Serine-8 Phosphorylation.

Post-translational modifications (PTMs) of amyloid-ß (Aß) species are implicated in the modulation of overall toxicities and aggregation propensities. We investigated the internal dynamics in the hydrophobic core of the truncated ΔE3 mutant fibrils of Aß1-40 and compared them with prior and new data for wild-type fibrils as well as with phosphorylated S8 fibrils. Deuteron static solid-state NMR techniques, spanning line-shape analysis, longitudinal relaxation, and chemical exchange saturation transfer methods, were employed to assess the rotameric jumps of several methyl-bearing and aromatic groups in the core of the fibrils. Taken together, the results indicate the rather significant influence of the PTMs on the hydrophobic core dynamics, which propagates far beyond the local site of the chemical modification. The phosphorylated S8 fibrils display an overall rigidifying of the core based on the higher activation barriers of motions than the wild-type fibrils, whereas the ΔE3 fibrils induce a broader variety of changes, some of which are thermodynamic in nature rather than the kinetic ones.

Molecular structure of an N-terminal phosphorylated ß-amyloid fibril.

The structural polymorphism in ß-amyloid (Aß) plaques from Alzheimer disease (AD) has been recognized as an important pathological factor. Plaques from sporadic AD patients contain fibrillar deposits of various amyloid proteins/peptides, including posttranslational modified Aß (PTM-Aß) subtypes. Although many PTM-Aßs were shown to accelerate the fibrillation process, increase neuronal cytotoxicity of aggregates, or enhance the stability of fibrils, the contribution of PTM-Aßs to structural polymorphisms and their pathological roles remains unclear. We report here the NMR-based structure for the Ser-8-phosphorylated 40-residue Aß (pS8-Aß40) fibrils, which shows significant difference to the wild-type fibrils, with higher cross-seeding efficiency and thermodynamic stability. Given these physicochemical properties, the structures originated from pS8-Aß40 fibrils may potentially dominate the polymorphisms in the mixture of wild-type and phosphorylated Aß deposits. Our results imply that Aß subtypes with "seeding-prone" properties may influence the polymorphisms of amyloid plaques through the cross-seeding process.

Deuterium solid-state NMR quadrupolar order rotating frame relaxation with applications to amyloid-ß fibrils.

We describe a new method for measuring molecular dynamics based on the deuterium solid-state nuclear magnetic resonance (NMR) quadrupolar order rotating frame relaxation rate R1ρ,Q under static conditions. The observed quadrupolar order coherence is created using the broad-band Jeener-Broekaert excitation and is locked with a weak radio frequency (RF) field. We describe the experimental and theoretical approaches and show applications to a selectively deuterated valine side chain of the phosphorylated amyloid-ß (1-40) fibrils phosphorylated at the serine-8 position. The R1ρ,Q rate is sensitive to the rotameric exchange mode. For biological samples, the low spin-lock field in the 5- to 10-kHz range has the advantage of avoiding sample heating and dehydration. Thus, it provides an alternative to approaches based on single-quantum coherence, which require larger spin-lock fields.

Solid-state NMR reveals a comprehensive view of the dynamics of the flexible, disordered N-terminal domain of amyloid-ß fibrils.

Amyloid fibril deposits observed in Alzheimer's disease comprise amyloid-ß (Aß) protein possessing a structured hydrophobic core and a disordered N-terminal domain (residues 1-16). The internal flexibility of the disordered domain is likely essential for Aß aggregation. Here, we used 2H static solid-state NMR methods to probe the dynamics of selected side chains of the N-terminal domain of Aß1-40 fibrils. Line shape and relaxation data suggested a two-state model in which the domain's free state undergoes a diffusive motion that is quenched in the bound state, likely because of transient interactions with the structured C-terminal domain. At 37 °C, we observed freezing of the dynamics progressively along the Aß sequence, with the fraction of the bound state increasing and the rate of diffusion decreasing. We also found that without solvation, the diffusive motion is quenched. The solvent acted as a plasticizer reminiscent of its role in the onset of global dynamics in globular proteins. As the temperature was lowered, the fraction of the bound state exhibited sigmoidal behavior. The midpoint of the freezing curve coincided with the bulk solvent freezing for the N-terminal residues and increased further along the sequence. Using 2H R1ρ measurements, we determined the conformational exchange rate constant between the free and bound states under physiological conditions. Zinc-induced aggregation leads to the enhancement of the dynamics, manifested by the faster conformational exchange, faster diffusion, and lower freezing-curve midpoints.

Deuteron Quadrupolar Chemical Exchange Saturation Transfer (Q-CEST) Solid-State NMR for Static Powder Samples: Approach and Applications to Amyloid-ß Fibrils.

We provide an experimental and computational framework for 2 H quadrupolar chemical exchange saturation transfer NMR experiments (Q-CEST) under static solid-state conditions for the quantification of dynamics on µs-ms timescales. Simulations using simple 2-site exchange models provide insights into the relation between spin dynamics and motions. Biological applications focus on two sites of amyloid-ß fibrils in the 3-fold symmetric polymorph. The first site, the methyl group of A2 of the disordered N-terminal domain, undergoes diffusive motions and conformational exchange due to transient interactions. Earlier 2 H rotating frame relaxation and quadrupolar CPMG measurements are combined with the Q-CEST approach to characterize the multiple conformational states of the domain. The second site, the methyl group of M35, spans the water-accessible cavity inside the fibrils' core and undergoes extensive rotameric exchange. Q-CEST permits us to refine the rotameric exchange model for this site and allows the more precise determination of populations and rotameric exchange rate constants than line shape analysis.

Effect of Post-Translational Modifications and Mutations on Amyloid-ß Fibrils Dynamics at N Terminus.

We investigate the variability in the dynamics of the disordered N-terminal domain of amyloid-ß fibrils (Aß), comprising residues 1-16 of Aß1-40, due to post-translational modifications and mutations in the ß-bend regions known to modulate aggregation properties. Using 2H static solid-state NMR approaches, we compare the dynamics in the wild-type Aß fibrils in the threefold symmetric polymorph with the fibrils from three post-translational modification sequences: isoaspartate-D7, the phosphorylation of S8, and an N-terminal truncation ΔE3. Additional comparisons are made with the mutants in the ß-bend region (residues 21-23) corresponding to the familial Osaka E22Δ deletion and D23N Iowa mutation. We also include the aggregates induced by Zn2+ ions. The dynamics are probed at the F4 and G9 positions. The main motional model involves two free states undergoing diffusion and conformational exchanges with the bound state in which the diffusion is quenched because of transient interactions involving fibril core and other intrastrand contacts. The fraction of the bound state increases in a sigmoidal fashion with a decrease in temperature. There is clear variability in the dynamics: the phosphorylation of S8 variant is the most rigid at the G9 site in line with structural studies, the ΔE3 fibrils are more flexible at the G9 site in line with the morphological fragmentation pattern, the Zn-induced aggregates are the most mobile, and the two ß-bend mutants have the strongest changes at the F4 site toward higher rigidity. Overall, the changes underlie the potential role of conformational ensembles in setting the stage for aggregation-prone states.

Deuterium Rotating Frame NMR Relaxation Measurements in the Solid State under Static Conditions for Quantification of Dynamics.

The feasibility of static deuterium rotating frame NMR relaxation measurements for characterization of slow timescale motions in powder systems is demonstrated. Using a model compound dimethyl sulfone-d6 , we show that these measurements yield conformational exchange rates and activation energy values in accordance with results obtained with other techniques. Furthermore, we demonstrate that the full Liouvillian approach as opposed to the Redfield approximation is necessary to analyze the experimental data.

Deuteron Solid-State NMR Relaxation Measurements Reveal Two Distinct Conformational Exchange Processes in the Disordered N-Terminal Domain of Amyloid-ß Fibrils.

We employed deuterium solid-state NMR techniques under static conditions to discern the details of the µs-ms timescale motions in the flexible N-terminal subdomain of Aß1-40 amyloid fibrils, which spans residues 1-16. In particular, we utilized a rotating frame (R1ρ ) and the newly developed time domain quadrupolar Carr-Purcell-Meiboom-Gill (QCPMG) relaxation measurements at the selectively deuterated side chains of A2, H6, and G9. The two experiments are complementary in terms of probing somewhat different timescales of motions, governed by the tensor parameters and the sampling window of the magnetization decay curves. The results indicated two mobile "free" states of the N-terminal domain undergoing global diffusive motions, with isotropic diffusion coefficients of 0.7-1 ⋅ 108 and 0.3-3 ⋅ 106 ad2 s-1 . The free states are also involved in the conformational exchange with a single bound state, in which the diffusive motions are quenched, likely due to transient interactions with the structured hydrophobic core. The conformational exchange rate constants are 2-3 ⋅ 105  s-1 and 2-3 ⋅ 104  s-1 for the fast and slow diffusion free states, respectively.

Basic experiments in 2H static NMR for the characterization of protein side-chain dynamics.

The focus of this review is the basic methodology for applications of static deuteron NMR for studies of dynamics in the side chains of proteins. We review experimental approaches for the measurements of static line shapes and relaxation rates as well as signal enhancement strategies using the multiple echo acquisition scheme. Further, we describe computational strategies for modeling jump and diffusive motions underlying experimental data. Applications are chosen from studies of amyloid fibrils comprising the amyloid-ß protein.

Correlated motions of C'-N and Cα-Cß pairs in protonated and per-deuterated GB3.

We investigated correlated µs-ms time scale motions of neighboring 13C'-15N and 13Cα-13Cß nuclei in both protonated and perdeuterated samples of GB3. The techniques employed, NMR relaxation due to cross-correlated chemical shift modulations, specifically target concerted changes in the isotropic chemical shifts of the two nuclei associated with spatial fluctuations. Field-dependence of the relaxation rates permits identification of the parameters defining the chemical exchange rate constant under the assumption of a two-site exchange. The time scale of motions falls into the intermediate to fast regime (with respect to the chemical shift time scale, 100-400 s-1 range) for the 13C'-15N pairs and into the slow to intermediate regime for the 13Cα-13Cß pairs (about 150 s-1). Comparison of the results obtained for protonated and deuterated GB3 suggests that deuteration has a tendency to reduce these slow scale correlated motions, especially for the 13Cα-13Cß pairs.

Flexibility and Solvation of Amyloid-ß Hydrophobic Core.

Amyloid fibril deposits found in Alzheimer disease patients are composed of amyloid-ß (Aß) protein forming a number of hydrophobic interfaces that are believed to be mostly rigid. We have investigated the µs-ms time-scale dynamics of the intra-strand hydrophobic core and interfaces of the fibrils composed of Aß1-40 protein. Using solid-state (2)H NMR line shape experiments performed on selectively deuterated methyl groups, we probed the 3-fold symmetric and 2-fold symmetric polymorphs of native Aß as well as the protofibrils of D23N Iowa mutant, associated with an early onset of Alzheimer disease. The dynamics of the hydrophobic regions probed at Leu-17, Leu-34, Val-36, and Met-35 side chains were found to be very pronounced at all sites and in all polymorphs of Aß, with methyl axis motions persisting down to 230-200 K for most of the sites. The dominant mode of motions is the rotameric side chain jumps, with the Met-35 displaying the most complex multi-modal behavior. There are distinct differences in the dynamics among the three protein variants, with the Val-36 site displaying the most variability. Solvation of the fibrils does not affect methyl group motions within the hydrophobic core of individual cross-ß subunits but has a clear effect on the motions at the hydrophobic interface between the cross-ß subunits, which is defined by Met-35 contacts. In particular, hydration activates transitions between additional rotameric states that are not sampled in the dry protein. Thus, these results support the existence of water-accessible cavity recently predicted by molecular dynamics simulations and suggested by cryo-EM studies.

Comparative Dynamics of Methionine Side-Chain in FMOC-Methionine and in Amyloid Fibrils.

We compared the dynamics of key methionine methyl groups in the water-accessible hydrophobic cavity of amyloid fibrils and Fluorenylmethyloxycarbonyl-Methionine (FMOC-Met), which renders general hydrophobicity to the environment without the complexity of the protein. Met35 in the hydrated cavity was recently found to undergo a dynamical cross-over from the dominance of methyl rotations at low temperatures to the dominance of diffusive motion of methyl axis at high temperatures. Current results indicate that in FMOC-Met this cross-over is suppressed, similar to what was observed for the dry fibrils, indicating that hydration of the cavity is driving the onset of the dynamical transition.

Fast Motions of Key Methyl Groups in Amyloid-ß Fibrils.

Amyloid-ß (Aß) peptide is the major component of plaques found in Alzheimer's disease patients. Using solid-state 2H NMR relaxation performed on selectively deuterated methyl groups, we probed the dynamics in the threefold symmetric and twofold symmetric polymorphs of native Aß as well as the protofibrils of the D23N mutant. Specifically, we investigated the methyl groups of two leucine residues that belong to the hydrophobic core (L17 and L34) as well as M35 residues belonging to the hydrophobic interface between the cross-ß subunits, which has been previously found to be water-accessible. Relaxation measurements performed over 310-140 K and two magnetic field strengths provide insights into conformational variability within and between polymorphs. Core packing variations within a single polymorph are similar to what is observed for globular proteins for the core residues, whereas M35 exhibits a larger degree of variability. M35 site is also shown to undergo a solvent-dependent dynamical transition in which slower amplitude motions of methyl axes are activated at high temperature. The motions, modeled as a diffusion of methyl axis, have activation energy by a factor of 2.7 larger in the twofold compared with the threefold polymorph, whereas D23N protofibrils display a value similar to the threefold polymorph. This suggests enhanced flexibility of the hydrophobic interface in the threefold polymorph. This difference is only observed in the hydrated state and is absent in the dry fibrils, highlighting the role of solvent at the cavity. In contrast, the dynamic behavior of the core is hydration-independent.

Restricted diffusion of methyl groups in proteins revealed by deuteron NMR: manifestation of intra-well dynamics.

The three-site hops of methyl groups are usually used as an approximation of the mechanistic description of motions responsible for the longitudinal NMR relaxation. Distinguishing between three-site hops and a more realistic mechanism of diffusion in a potential requires extended experimental and computational analysis. In order to achieve this goal, in this work the restricted diffusion is decomposed into two independent modes, namely, the jumps between potential wells and intra-well fluctuations, assuming time scale separation between these modes. This approach allows us to explain the rise in the theoretical value of T1 minimum for the restricted diffusion mechanism compared with the three-site hops mechanism via rescaling the three-site hops correlation function by the order parameter of intra-well motions. The main result of the paper is that, in general, intra-well dynamics can be visible in NMR even in the limit of large barrier heights in contrast to the common view that this limit converges to the three-site hops mechanism. Based on a previously collected detailed set of deuteron NMR relaxation and spectral data in the villin headpiece subdomain protein over a wide temperature range of 300-31 K, we are then able to conclude that the mechanism of diffusion in the threefold potential is likely to be the main source of the dynamics in this system.

Surgical Approaches to Petrous Apex Cholesterol Granulomas: A Systematic Review and Network Meta-analysis.

OBJECTIVE: To compare the outcomes of different surgical approaches to petrous apex cholesterol granulomas (PACG). DATA SOURCES: PubMed, Embase, Google Scholar, Cochrane, and Web of Science. REVIEW METHODS: Following the Preferred Reporting Items for Systematic Reviews and Meta-analyses-Network Meta-analyses guidelines, databases were searched from inception to November 31, 2022. Studies comparing two or more approaches were included. Reviews and population studies were excluded. The main outcome measures were the resolution of symptoms, serviceable hearing, complication, and revision rates. RESULTS: The search yielded 2132 studies. After applying inclusion and exclusion criteria, 15 studies remained, consisting of 214 patients treated with lateral approaches (n = 182) or anterior endonasal approaches (n = 32). The efficacy of lateral and anterior endonasal approaches in achieving symptom resolution was comparable (73% vs. 68%, p = 0.5). Both exhibited similar rates of complications (33% vs. 37%, p = 0.3), albeit with distinct profiles. Lateral approaches were associated with higher rates of facial palsy and sensorineural hearing loss (44% vs. 18%, p = 0.03). Anterior endonasal approaches demonstrated higher rates of epistaxis and cerebrospinal fluid leak (15% vs. 1%, p = 0.001). Anterior endonasal approaches exhibited lower revision rates (OR: 0.35, 95% CI: 0.14-0.88). The placement of a stent in both approaches was associated with higher symptom resolution (OR: 5.12, 95% CI: 1.05-9.97) and lower revision rates (OR: 0.71, 95% CI: 0.33-0.92). CONCLUSIONS: Anterior endonasal approaches yield lower revision rates compared to lateral approaches for PACG. Both approaches demonstrate similar effectiveness in symptom resolution and comparable rates of complications, with distinct profiles. Facial nerve and hearing status are important factors that should be addressed when selecting the approach. Stenting is beneficial. LEVEL OF EVIDENCE: NA Laryngoscope, 134:1540-1550, 2024.