How important is the N-terminal acetylation of alpha-synuclein for its function and aggregation into amyloids?

N-α-acetylation is a frequently occurring post-translational modification in eukaryotic proteins. It has manifold physiological consequences on the regulation and function of several proteins, with emerging studies suggesting that it is a global regulator of stress responses. For decades, in vitro biochemical investigations into the precise role of the intrinsically disordered protein alpha-synuclein (αS) in the etiology of Parkinson's disease (PD) were performed using non-acetylated αS. The N-terminus of α-synuclein is now unequivocally known to be acetylated in vivo, however, there are many aspects of this post-translational modifications that are not understood well. Is N-α-acetylation of αS a constitutive modification akin to most cellular proteins, or is it spatio-temporally regulated? Is N-α-acetylation of αS relevant to the as yet elusive function of αS? How does the N-α-acetylation of αS influence the aggregation of αS into amyloids? Here, we provide an overview of the current knowledge and discuss prevailing hypotheses on the impact of N-α-acetylation of αS on its conformational, oligomeric, and fibrillar states. The extent to which N-α-acetylation of αS is vital for its function, membrane binding, and aggregation into amyloids is also explored here. We further discuss the overall significance of N-α-acetylation of αS for its functional and pathogenic implications in Lewy body formation and synucleinopathies.

Minimally Invasive Segmental Artery Coil Embolization (MISACE) Prior to Endovascular Thoracoabdominal Aortic Aneurysm Repair.

PURPOSE: Minimally Invasive Segmental Artery Coil Embolization (MISACE) is a novel approach to reduce paraplegia risk in Thoracoabdominal aortic aneurysm (TAAA) repair with limited data. We report our experience with MISACE as a method of spinal cord pre-conditioning to prevent spinal cord ischemia following endovascular repair of TAAA. MATERIAL AND METHODS: A retrospective analysis of 17 patients who had an attempted MISACE prior to endovascular TAAA repair with mean follow-up of 350 days (2017-2020). Baseline patient and aneurysm characteristics along with procedural technique and outcomes were analyzed. RESULTS: Mean age of 69 years and 76.5% were males. TAAA Crawford classification were II, n = 6 (35.3%), III, n = 4 (23.5%) and IV, n = 5 (29.4%). The mean aortic diameter was 70.6 ± 10.9 mm. Staged repair was performed on 9 patients. Technically successful embolization occurred in 14 patients (82.4%) and was unsuccessful in 3 patients. The median number of embolized arteries was 3 and 71% of the target arteries were between T9 and T12. Mean fluoroscopy time was 51.5 ± 22.5 min and mean contrast volume used was 132.8 ± 56.1 mL. Average number of catheters used was 4.6 and 3.5 wires. No complications related to the procedure. Mean interval between embolization to endovascular TAAA repair was 51.2 days (5-110 days). All patients received spinal drainage at the time of repair. Postoperatively, 2/14 of patients developed paraparesis in the MISACE successful group and 1/3 patients developed paraplegia in the unsuccessful group. CONCLUSIONS: MISACE is a promising strategy to prevent SCI. This data demonstrates the technique is feasible and safe but anatomic challenges remain.

Orthostatic Myoclonic Jerks in a Case of Hashimoto's Encephalopathy.

Hashimoto's encephalopathy (HE) is an uncommon syndrome with the characteristic triad of positive antithyroid antibodies (most commonly antibodies to thyroid peroxidase), response to steroids, and clinical picture presenting either as stroke-like pattern of focal neurological deficit or slowly progressive cognitive impairment. Myoclonus or tremors, seizures, and psychosis are other associated features which can be seen in HE. Herein, we report a girl with an uncommon presentation of orthostatic axial and myoclonic jerks in bilateral lower limbs in a case of HE.

Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair.

Breast cancer type two susceptibility protein (BRCA2) is an essential protein in genome maintenance, homologous recombination (HR), and replication fork protection. Its function includes multiple interaction partners and requires timely localization to relevant sites in the nucleus. We investigated the importance of the highly conserved DNA-binding domain (DBD) and C-terminal domain (CTD) of BRCA2. We generated BRCA2 variants missing one or both domains in mouse embryonic stem (ES) cells and defined their contribution in HR function and dynamic localization in the nucleus, by single-particle tracking of BRCA2 mobility. Changes in molecular architecture of BRCA2 induced by binding partners of purified BRCA2 were determined by scanning force microscopy. BRCA2 mobility and DNA-damage-induced increase in the immobile fraction were largely unaffected by C-terminal deletions. The purified proteins missing CTD and/or DBD were defective in architectural changes correlating with reduced HR function in cells. These results emphasize BRCA2 activity at sites of damage beyond promoting RAD51 delivery.

Conformational flexibility and oligomerization of BRCA2 regions induced by RAD51 interaction.

BRCA2 is a key breast cancer associated protein that is predicted to have interspersed regions of intrinsic disorder. Intrinsic disorder coupled with large size likely allows BRCA2 to sample a broad range of conformational space. We expect that the resulting dynamic arrangements of BRCA2 domains are a functionally important aspect of its role in homologous recombination DNA repair. To determine the architectural organization and the associated conformational landscape of BRCA2, we used scanning force microscopy based single molecule analyses to map the flexible regions of the protein and characterize which regions influence oligomerization. We show that the N- and the C-terminal regions are the main flexible regions. Both of these regions also influence BRCA2 oligomerization and interaction with RAD51. In the central Brc repeat region, Brc 1-4 and Brc 5-8 contribute synergistically to BRCA2 interaction with RAD51. We also analysed several single amino acid changes that are potentially clinically relevant and found one, the variant of F1524V, which disrupts key interactions and alters the conformational landscape of the protein. We describe the overall conformation spectrum of BRCA2, which suggests that dynamic structural transitions are key features of its biological function, maintaining genomic stability.

Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils.

Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semi-quantitatively in vitro, specifically probing the fibrillar cross-ß-sheet content. In recent years, structural polymorphism of amyloid fibrils has been shown to be an important aspect of amyloid formation, both in vitro and in neurodegenerative diseases. Therefore, understanding ThT-amyloid interactions in the context of structural polymorphism of amyloids is necessary for correct interpretation of ThT fluorescence data. Here we study the influence of fibril morphology on ThT fluorescence and ThT binding sites, with two morphologically distinct but chemically identical α-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behaviour although their ß-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibril-bound ThT reveal the presence of at least two qualitatively different ThT binding sites on the polymorphs. The relative distributions of the binding sites on the fibril surfaces appear to be morphology dependent, thus determining the observed polymorph-specific ThT fluorescence intensities. These results, highlighting the role of fibril morphology in ThT-based amyloid studies, underline the relevance of polymorphs in ThT-amyloid interaction and can explain the variability often observed in ThT amyloid binding assays.

The Recombination Mediator BRCA2: Architectural Plasticity of Recombination Intermediates Revealed by Single-Molecule Imaging (SFM/TIRF).

Cellular functions are defined by dynamic assembly, rearrangement, and disassembly of biomolecules to achieve control and specificity. As an example, effective DNA repair is brought about by the concerted action of several DNA processing proteins. Both changes in the structure of individual proteins and in the arrangement of multiple proteins together (referred to here as architecture) are inherent to biological function. These dynamic changes are exemplified in the breast cancer susceptibility protein 2 (BRCA2). BRCA2 is a DNA repair protein that undergoes changes in its own structure and affects changes in molecular architecture with partners during homologous recombination (HR) repair of DNA double strand breaks. These challenging features of BRCA2 protein, its size and predicted stretches of intrinsically disordered regions, have made it difficult to determine the structural consequences and mechanistic importance of interactions between full-length BRCA2 with RAD51 and other HR proteins. In this chapter, we describe scanning force microscopy (SFM)-based approaches to study DNA-protein complexes involved in HR, the architectural plasticity of full-length BRCA2, and the dynamic reorganization of these molecular components associated with essential steps of HR.

Distinct Mechanisms Determine α-Synuclein Fibril Morphology during Growth and Maturation.

Amyloid polymorphs have become one of the focal points of molecular studies of neurodegenerative diseases like Parkinson's disease. Due to their distinct biochemical properties and prion-like characteristics, insights into the molecular origin and stability of amyloid polymorphs over time are crucial for understanding the potential role of amyloid polymorphism in these diseases. Here, we systematically study the fibrillization of recombinantly produced human α-synuclein (αSyn) over an extended period of time to unravel the origin and temporal evolution of polymorphism. We follow morphological changes in the same fibril sample with atomic force microscopy over a period of 1 year. We show that wild-type (wt) αSyn fibrils undergo a slow maturation over time after reaching the plateau phase of aggregation (as detected in a Thioflavin-T fluorescence assay). This maturation, visualized by changes in the fibril periodicity over time, is absent in the disease mutant fibrils. The ß-sheet content of the plateau phase and matured fibrils, obtained using Fourier transform infrared spectroscopy, is however similar for the αSyn protein sequences, suggesting that the morphological changes in wt αSyn fibrils are tertiary or quaternary in origin. Furthermore, results from a reversibility assay show that the plateau phase fibrils do not disassemble over time. Together, the observed changes in the periodicity distributions and stability of the fibrillar core over time point toward two distinct mechanisms that determine the morphology of wt αSyn fibrils: competitive growth between different polymorphs during the fibrillization phase followed by a process wherein fibrils undergo slow maturation or annealing.

Does Technical Success of Angioplasty in Dysfunctional Hemodialysis Accesses Correlate with Access Patency?

PURPOSE: To study if <30 % residual stenosis post angioplasty (PTA) correlates with primary access circuit patency, and if any variables predict technical success. MATERIALS AND METHODS: A prospective observational study was performed between January 2009 and December 2012, wherein 76 patients underwent 154 PTA events in 56 prosthetic grafts (AVG) and 98 autogenous fistulas (AVF). Data collected included patient age, gender, lesion location and laterality, access type and location, number of prior interventions, and transonic flow rates pre- and postintervention. Impact of technical outcome on access patency was assessed. Univariate logistic regression was used to assess the impact of variables on technical success with significant factors assessed with a multiple variable model. RESULTS: Technical success rates of PTA in AVFs and AVGs were 79.6 and 76.7 %, respectively. Technical failures of PTA were associated with an increased risk of patency loss among circuits with AVFs (p < 0.05), but not with AVGs (p = 0.7). In AVFs, primary access patency rates between technical successes and failures at three and 6 months were 74.4 versus 61.9 % (p = 0.3) and 53.8 versus 23.8 % (p < 0.05), respectively. In AVGs, primary access patency rates between technical successes and failures at three and six months were 72.1 versus 53.9 % (p = 0.5) and 33.6 versus 38.5 % (p = 0.8), respectively. Transonic flow rates did not significantly differ among technically successful or failed outcomes at one or three months. CONCLUSION: Technical failures of PTA had a significant impact on access patency among AVFs with a trend toward poorer access patency within AVGs.

Functionally different α-synuclein inclusions yield insight into Parkinson's disease pathology.

The formation of α-synuclein (α-S) amyloid aggregates, called Lewy bodies (LBs), is a hallmark of Parkinson's disease (PD). The function of LBs in the disease process is however still unclear; they have been associated with both neuroprotection and toxicity. To obtain insight into this contradiction, we induced the formation of α-S inclusions, using three different induction methods in SH-SY5Y cells and rat-derived primary neuronal cells. Using confocal and STED microscopy we observed induction-dependent differences in α-S inclusion morphology, location and function. The aggregation of α-S in functionally different compartments correlates with the toxicity of the induction method measured in viability assays. The most cytotoxic treatment largely correlates with the formation of proteasome-associated, juxta-nuclear inclusions. With less toxic methods cytosolic deposits that are not associated with the proteasome are more prevalent. The distribution of α-S over at least two different types of inclusions is not limited to cell models, but is also observed in primary neuronal cells and in human mesencephalon. The existence of functionally different LBs, in vivo and in vitro, gives important insights in the impact of Lewy Body formation on neuronal functioning and may thereby provide a platform for discovering therapeutics.

Conformational Compatibility Is Essential for Heterologous Aggregation of α-Synuclein.

Under aggregation-prone conditions, soluble amyloidogenic protein monomers can self-assemble into fibrils or they can fibrillize on preformed fibrillar seeds (seeded aggregation). Seeded aggregations are known to propagate the morphology of the seeds in the event of cross-seeding. However, not all proteins are known to cross-seed aggregation. Cross-seeding has been proposed to be restricted either because of differences in the protein sequences or because of conformations between the seeds and the soluble monomers. Here, we examine cross-seeding efficiency between three α-synuclein sequences, wild-type, A30P, and A53T, each varying in only one or two amino acids but forming morphologically distinct fibrils. Results from bulk Thioflavin-T measurements, monomer incorporation quantification, single fibril fluorescence microscopy, and atomic force microscopy show that under the given solution conditions conformity between the conformation of seeds and monomers is essential for seed elongation. Moreover, elongation characteristics of the seeds are defined by the type of seed.

Elastic Recoil after Balloon Angioplasty in Hemodialysis Accesses: Does It Actually Occur and Is It Clinically Relevant?

Purpose To qualify and quantify elastic recoil and determine its effect on access patency. Materials and Methods Research ethics board approval was obtained and all patients signed an informed consent form. This was a prospective, nonrandomized study of mature accesses that underwent balloon percutaneous transluminal angioplasty (PTA) between January 2009 and December 2012. After PTA, completion fistulography was performed at 0-, 5-, 10-, and 15-minute intervals. From Digital Imaging and Communications in Medicine images, percentage of lesion stenosis before and after PTA was measured at each time point. A total of 76 patients (44 men, 32 women; mean age, 59.6 years) were enrolled and underwent 154 PTAs in 56 grafts and 98 fistulas. Venous elastic recoil was defined as recurrent luminal narrowing greater than 50% within 15 minutes after full effacement of the stenosis by the angioplasty balloon. Data collected included sex, age, access type and location, lesion location, length, and time to next intervention. Access patency was estimated by using Kaplan-Meier survival method, association of variables with the risk of loss of patency was assessed by using a Cox proportional hazards model, and a multiple variable model was examined by considering all variables. Results Technical success of PTA with less than 30% residual stenosis was 78%. By 15 minutes, 15.6% (24 of 154) of treated lesions recurrently narrowed by more than 50%, with a majority observed at 5 minutes (15 of 24). Technical failure of PTA was predictive of elastic recoil (P < .001), as was cephalic arch stenosis in fistulas (P = .047) and autogenous fistulas (P = .04). Elastic recoil, when it did occur, did not influence patency. Six-month primary patency was 34.8% in grafts and 47.1% in fistulas. Conclusion Venous elastic recoil after PTA of stenoses in hemodialysis access circuits is common, but its occurrence does not influence access primary patency after PTA. (©) RSNA, 2015.

Solution conditions define morphological homogeneity of α-synuclein fibrils.

The intrinsically disordered human α-synuclein (αSyn) protein exhibits considerable heterogeneity in in vitro fibrillization reactions. Using atomic force microscopy (AFM) we show that depending on the solvent conditions, A140C mutant and wild-type αSyn can be directed to reproducibly form homogeneous populations of fibrils exhibiting regular periodicity. Results from Thioflavin-T fluorescence assays, determination of residual monomer concentrations and native polyacrylamide gel electrophoresis reveal that solvent conditions including EDTA facilitate incorporation of a larger fraction of monomers into fibrils. The fibrils formed in 10mM Tris-HCl, 10mM NaCl and 0.1mM EDTA at pH7.4 display a narrow distribution of periodicities with an average value of 102±6nm for the A140C mutant and 107±9nm for wt αSyn. The ability to produce a homogeneous fibril population can be instrumental in understanding the detailed structural features of fibrils and the fibril assembly process. Moreover, the availability of morphologically well-defined fibrils will enhance the potential for use of amyloids as biological nanomaterials.

Improving pediatric radiology in India.

BACKGROUND: India had a population of 1,259,271,000 in 2012, with 31% of its population composed of children younger than 15 years. In comparison, children younger than 15 compose 20% of the population in the United States and 16% in Canada. Despite the differences in this demographic, little emphasis is placed on pediatric radiology in India. OBJECTIVE: To conduct a needs assessment evaluating the adequacy of pediatric radiology in India and to establish a pediatric radiology education program in India. MATERIALS AND METHODS: We developed a questionnaire to assess radiologists' perspective on the quality of training in pediatric radiology. Responses were obtained from attendees at a pediatric radiology education program in Mumbai. These data were used to obtain funding and implement a program to increase awareness and stimulate pediatric radiology training at select institutions across India. RESULTS: Out of 86 respondents, 82% indicated that their academic institutions did not place emphasis on dedicated pediatric radiology training, and 63% indicated they received less than 2 weeks of dedicated training. Of the respondents, 77% said their institutions practiced pediatric radiology with inadequate standard of care and 75% of respondents said pediatric radiology would gain increasing importance in the future. Outcomes of the implemented program included increased awareness of pediatric radiology and establishment of a pediatric radiology fellowship program in India. CONCLUSION: Education and training in pediatric radiology in India is inadequate. Focused initiatives have the potential to improve the standards set for pediatric radiology in India. Similar initiatives could help develop pediatric radiology in other developing countries.

A hydrogen bond regulates slow motions in ubiquitin by modulating a ß-turn flip.

Proteins exist as conformational ensembles composed of multiple interchanging substates separated by kinetic barriers. Interconverting conformations are often difficult to probe, owing to their sparse population and transient nature. Here, we report the identification and characterization of a subset of conformations in ubiquitin that participate in microsecond-to-millisecond motions in the amides of Ile23, Asn25, and Thr55. A novel side chain to the backbone hydrogen bond that regulates these motions has also been identified. Combining our NMR studies with the available X-ray data, we have unearthed the physical process underlying slow motions-the interconversion of a type I into a type II ß-turn flip at residues Glu51 through Arg54. Interestingly, the dominant conformer of wild-type ubiquitin observed in solution near neutral pH is only represented by about 22% of the crystal structures. The conformers generated as a result of the dynamics of the hydrogen bond appear to be correlated to ligand recognition by ubiquitin.