The alpha-synuclein oligomers activate nuclear factor of activated T-cell (NFAT) modulating synaptic homeostasis and apoptosis.

BACKGROUND: Soluble oligomeric forms of alpha-synuclein (aSyn-O) are believed to be one of the main toxic species in Parkinson's disease (PD) leading to degeneration. aSyn-O can induce Ca2+ influx, over activating downstream pathways leading to PD phenotype. Calcineurin (CN), a phosphatase regulated by Ca2+ levels, activates NFAT transcription factors that are involved in the regulation of neuronal plasticity, growth, and survival. METHODS: Here, using a combination of cell toxicity and gene regulation assays performed in the presence of classical inhibitors of the NFAT/CN pathway, we investigate NFAT's role in neuronal degeneration induced by aSyn-O. RESULTS: aSyn-O are toxic to neurons leading to cell death, loss of neuron ramification and reduction of synaptic proteins which are reversed by CN inhibition with ciclosporin-A or VIVIT, a NFAT specific inhibitor. aSyn-O induce NFAT nuclear translocation and transactivation. We found that aSyn-O modulates the gene involved in the maintenance of synapses, synapsin 1 (Syn 1). Syn1 mRNA and protein and synaptic puncta are drastically reduced in cells treated with aSyn-O which are reversed by NFAT inhibition. CONCLUSIONS: For the first time a direct role of NFAT in aSyn-O-induced toxicity and Syn1 gene regulation was demonstrated, enlarging our understanding of the pathways underpinnings synucleinopathies.

Disease-associated mutations impacting BC-loop flexibility trigger long-range transthyretin tetramer destabilization and aggregation.

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the extracellular deposition of the transport protein transthyretin (TTR) as amyloid fibrils. Despite the progress achieved in recent years, understanding why different TTR residue substitutions lead to different clinical manifestations remains elusive. Here, we studied the molecular basis of disease-causing missense mutations affecting residues R34 and K35. R34G and K35T variants cause vitreous amyloidosis, whereas R34T and K35N mutations result in amyloid polyneuropathy and restrictive cardiomyopathy. All variants are more sensitive to pH-induced dissociation and amyloid formation than the wild-type (WT)-TTR counterpart, specifically in the variants deposited in the eyes amyloid formation occurs close to physiological pHs. Chemical denaturation experiments indicate that all the mutants are less stable than WT-TTR, with the vitreous amyloidosis variants, R34G and K35T, being highly destabilized. Sequence-induced stabilization of the dimer-dimer interface with T119M rendered tetramers containing R34G or K35T mutations resistant to pH-induced aggregation. Because R34 and K35 are among the residues more distant to the TTR interface, their impact in this region is therefore theorized to occur at long range. The crystal structures of double mutants, R34G/T119M and K35T/T119M, together with molecular dynamics simulations indicate that their strong destabilizing effect is initiated locally at the BC loop, increasing its flexibility in a mutation-dependent manner. Overall, the present findings help us to understand the sequence-dynamic-structural mechanistic details of TTR amyloid aggregation triggered by R34 and K35 variants and to link the degree of mutation-induced conformational flexibility to protein aggregation propensity.

Extracellular alpha-synuclein: Sensors, receptors, and responses.

Synucleinopathies are a group of progressive neurodegenerative diseases known for the accumulation of insoluble aggregates containing the protein alpha-synuclein (aSyn). Recently, it has been assumed that pathology spreads in the brain during disease progression, implying that, at some point in the process, aSyn may exist outside of cells. In this context, extracellular-aSyn (e-aSyn) might transduce signals to the inside of the cells it interacts with, and/or be internalized by different types of cells through the extracellular matrix. Both negatively charged lipids and membrane receptors have been hypothesized as modulators of the loss of cellular homeostasis and cytotoxicity, and of the internalization of e-aSyn. Internalized e-aSyn causes the disruption of multiple cellular processes such as the autophagy lysosomal pathway (ALP), mitochondrial function, endoplasmic reticulum (ER)-stress, UPR activation, or vesicular transport. These processes happen not only in neurons but also in glial cells, activating inflammatory or anti-inflammatory pathways that can affect both neuronal function and survival, thereby affecting disease progression. In this review, we explore possible effects e-aSyn, all the way from the extracellular matrix to the nucleus. In particular, we highlight the glial-neuronal relationship as this is particularly relevant in the context of the spreading of aSyn pathology in synucleinopathies.

Padded Headgear does not Reduce the Incidence of Match Concussions in Professional Men's Rugby Union: A Case-control Study of 417 Cases.

Concussion is the most common match injury in rugby union. Some players wear padded headgear, but whether this protects against concussion is unclear. In professional male rugby union players, we examined: (i) the association between the use of headgear and match concussion injury incidence, and (ii) whether wearing headgear influenced time to return to play following concussion. Using a nested case-control within a cohort study, four seasons (2013-2017) of injury data from 1117 players at the highest level of rugby union in England were included. Cases were physician-diagnosed concussion injuries. Controls were other contact injuries (excluding all head injuries). We determined headgear use by viewing video footage. Sixteen percent of cases and controls wore headgear. Headgear use had no significant effect on concussion injury incidence (adjusted odds ratio=1.05, 95% CI: 0.71-1.56). Median number of days absent for concussion whilst wearing headgear was 8 days, compared with 7 days without headgear. Having sustained a concussion in the current or previous season increased the odds of concussion more than four-fold (odds ratio=4.55, 95% CI: 3.77-5.49). Wearing headgear was not associated with lower odds of concussions or a reduced number of days' absence following a concussion.

Physical Demands of Refereeing Rugby Sevens Matches at Different Competitive Levels.

ABSTRACT: Sant'Anna, RT, Roberts, SP, Moore, LJ, and Stokes, KA. Physical demands of refereeing rugby sevens matches at different competitive levels. J Strength Cond Res 35(11): 3164-3169, 2021-The aim of this study was to compare the physical demands of officiating across different competitive levels in rugby sevens. An observational design was used involving 27 referees (26 men and 1 woman, age: 27 ± 6 years, body mass [mean ± SD]: 78.5 ± 9.3 kg, and height: 179 ± 5 cm). Global Navigation Satellite Systems data were collected across a total of 114 matches during 5 separate rugby sevens tournaments played in England-between May and July 2018-categorized into 4 competitive levels: (a) international, (b) professional, (c) semiprofessional, and (d) amateur. Compared with referees officiating at the international, professional, and semiprofessional levels, referees officiating at the amateur level covered less total (p < 0.001) and relative distance (p < 0.001). In addition, these referees covered more distance walking and jogging (p < 0.001). Amateur referees also completed fewer sprints (p = 0.006), repeated high-intensity efforts (RHIEs) per game (p < 0.001), and spent longer between RHIEs (p = 0.015). Finally, for the amateur referees, the duration of the longest repeated high-intensity bout (i.e., worst case scenario) was lower (p < 0.001), with less distance covered (p < 0.001) and fewer high-intensity accelerations (p < 0.001). Refereeing rugby sevens is therefore more physically demanding at higher competitive levels, particularly in terms of high-intensity efforts. The results provide vital information for practitioners involved in the physical preparation of rugby sevens referees.

Environmental and genetic factors support the dissociation between α-synuclein aggregation and toxicity.

Synucleinopathies are a group of progressive disorders characterized by the abnormal aggregation and accumulation of α-synuclein (aSyn), an abundant neuronal protein that can adopt different conformations and biological properties. Recently, aSyn pathology was shown to spread between neurons in a prion-like manner. Proteins like aSyn that exhibit self-propagating capacity appear to be able to adopt different stable conformational states, known as protein strains, which can be modulated both by environmental and by protein-intrinsic factors. Here, we analyzed these factors and found that the unique combination of the neurodegeneration-related metal copper and the pathological H50Q aSyn mutation induces a significant alteration in the aggregation properties of aSyn. We compared the aggregation of WT and H50Q aSyn with and without copper, and assessed the effects of the resultant protein species when applied to primary neuronal cultures. The presence of copper induces the formation of structurally different and less-damaging aSyn aggregates. Interestingly, these aggregates exhibit a stronger capacity to induce aSyn inclusion formation in recipient cells, which demonstrates that the structural features of aSyn species determine their effect in neuronal cells and supports a lack of correlation between toxicity and inclusion formation. In total, our study provides strong support in favor of the hypothesis that protein aggregation is not a primary cause of cytotoxicity.

The importance of a gatekeeper residue on the aggregation of transthyretin: implications for transthyretin-related amyloidoses.

Protein aggregation into ß-sheet-enriched amyloid fibrils is associated with an increasing number of human disorders. The adoption of such amyloid conformations seems to constitute a generic property of polypeptide chains. Therefore, during evolution, proteins have adopted negative design strategies to diminish their intrinsic propensity to aggregate, including enrichment of gatekeeper charged residues at the flanks of hydrophobic aggregation-prone segments. Wild type transthyretin (TTR) is responsible for senile systemic amyloidosis, and more than 100 mutations in the TTR gene are involved in familial amyloid polyneuropathy. The TTR 26-57 segment bears many of these aggressive amyloidogenic mutations as well as the binding site for heparin. We demonstrate here that Lys-35 acts as a gatekeeper residue in TTR, strongly decreasing its amyloidogenic potential. This protective effect is sequence-specific because Lys-48 does not affect TTR aggregation. Lys-35 is part of the TTR basic heparin-binding motif. This glycosaminoglycan blocks the protective effect of Lys-35, probably by neutralization of its side chain positive charge. A K35L mutation emulates this effect and results in the rapid self-assembly of the TTR 26-57 region into amyloid fibrils. This mutation does not affect the tetrameric protein stability, but it strongly increases its aggregation propensity. Overall, we illustrate how TTR is yet another amyloidogenic protein exploiting negative design to prevent its massive aggregation, and we show how blockage of conserved protective features by endogenous factors or mutations might result in increased disease susceptibility.

Mammalian prion protein (PrP) forms conformationally different amyloid intracellular aggregates in bacteria.

BACKGROUND: An increasing number of proteins are being shown to assemble into amyloid structures that lead to pathological states. Among them, mammalian prions outstand due to their ability to transmit the pathogenic conformation, becoming thus infectious. The structural conversion of the cellular prion protein (PrP(C)), into its misfolded pathogenic form (PrP(Sc)) is the central event of prion-driven pathologies. The study of the structural properties of intracellular amyloid aggregates in general and of prion-like ones in particular is a challenging task. In this context, the evidence that the inclusion bodies formed by amyloid proteins in bacteria display amyloid-like structural and functional properties make them a privileged system to model intracellular amyloid aggregation. RESULTS: Here we provide the first demonstration that recombinant murine PrP and its C-terminal domain (90-231) attain amyloid conformations inside bacteria. Moreover, the inclusions formed by these two PrP proteins display conformational diversity, since they differ in fibril morphology, binding affinity to amyloid dyes, stability, resistance to proteinase K digestion and neurotoxicity. CONCLUSIONS: Overall, our results suggest that modelling PrP amyloid formation in microbial cell factories might open an avenue for a better understanding of the structural features modulating the pathogenic impact of this intriguing protein.

Inhibition of human transthyretin aggregation by non-steroidal anti-inflammatory compounds: a structural and thermodynamic analysis.

Transthyretin (TTR) is a homotetrameric protein that circulates in plasma and cerebral spinal fluid (CSF) whose aggregation into amyloid fibrils has been associated with at least two different amyloid diseases: senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP). In SSA aggregates are composed of WT-TTR, while in FAP more than 100 already-described variants have been found in deposits. Until now, TTR-related diseases have been untreatable, although a new drug called Tafamidis has been approved only in Europe to specifically treat V30M patients. Thus, new strategies are still necessary to treat FAP caused by other variants of TTR. TTR has two channels in the dimer interface that bind to the hormone thyroxin and that have been used to accommodate anti-amyloidogenic compounds. These compounds stabilize the tetramers, rendering TTR less amyloidogenic. Here, we investigated the effects of three non-steroidal anti-inflammatory compounds-sulindac (SUL), indomethacin (IND) and lumiracoxib (LUM)-as tetramer stabilizers and aggregation inhibitors. WT-TTR and the very aggressive TTR variant L55P were used as models. These compounds were able to stabilize TTR against high hydrostatic pressure (HHP), increasing the ΔGf by several kcal. They were also effective in inhibiting WT-TTR and L55P acid- or HHP-induced aggregation; in particular, LUM and IND were very effective, inhibiting almost 100% of the aggregation of both proteins under certain conditions. The species formed when aggregation was performed in the presence of these compounds were much less toxic to cells in culture. The crystal structures of WT-TTR bound to the three compounds were solved at high resolution, allowing the identification of the relevant protein:drug interactions. We discuss here the ligand-binding features of LUM, IND and SUL to TTR, emphasizing the critical interactions that render the protein more stable and less amyloidogenic.

Connection between MHC class II binding and aggregation propensity: The antigenic peptide 10 of Paracoccidioides brasiliensis as a benchmark study.

The aggregation of epitopes that are also able to bind major histocompatibility complex (MHC) alleles raises questions around the potential connection between the formation of epitope aggregates and their affinities to MHC receptors. We first performed a general bioinformatic assessment over a public dataset of MHC class II epitopes, finding that higher experimental binding correlates with higher aggregation-propensity predictors. We then focused on the case of P10, an epitope used as a vaccine candidate against Paracoccidioides brasiliensis that aggregates into amyloid fibrils. We used a computational protocol to design variants of the P10 epitope to study the connection between the binding stabilities towards human MHC class II alleles and their aggregation propensities. The binding of the designed variants was tested experimentally, as well as their aggregation capacity. High-affinity MHC class II binders in vitro were more disposed to aggregate forming amyloid fibrils capable of binding Thioflavin T and congo red, while low affinity MHC class II binders remained soluble or formed rare amorphous aggregates. This study shows a possible connection between the aggregation propensity of an epitope and its affinity for the MHC class II cleft.

Pentameric Thiophene as a Probe to Monitor EGCG's Remodeling Activity of Mature Amyloid Fibrils: Overcoming Signal Artifacts of Thioflavin T.

Thioflavin T fluorescence is a gold standard probe for the detection of amyloid fibrils. Herein, we showed that mature amyloid fibrils incubated with polyphenol epigallocatechin gallate (EGCG) present a fast reduction of the thioflavin T fluorescence, which is not related to remodeling activity. We propose the use of the pentameric thiophene fluorescence for monitoring the polyphenol remodeling activity.

Peptides derived from gp43, the most antigenic protein from Paracoccidioides brasiliensis, form amyloid fibrils in vitro: implications for vaccine development.

Fungal infection is an important health problem in Latin America, and in Brazil in particular. Paracoccidioides (mainly P. brasiliensis and P. lutzii) is responsible for paracoccidioidomycosis, a disease that affects mainly the lungs. The glycoprotein gp43 is involved in fungi adhesion to epithelial cells, which makes this protein an interesting target of study. A specific stretch of 15 amino acids that spans the region 181-195 (named P10) of gp43 is an important epitope of gp43 that is being envisioned as a vaccine candidate. Here we show that synthetic P10 forms typical amyloid aggregates in solution in very short times, a property that could hamper vaccine development. Seeds obtained by fragmentation of P10 fibrils were able to induce the aggregation of P4, but not P23, two other peptides derived from gp43. In silico analysis revealed several regions within the P10 sequence that can form amyloid with steric zipper architecture. Besides, in-silico proteolysis studies with gp43 revealed that aggregation-prone, P10-like peptides could be generated by several proteases, which suggests that P10 could be formed under physiological conditions. Considering our data in the context of a potential vaccine development, we redesigned the sequence of P10, maintaining the antigenic region (HTLAIR), but drastically reducing its aggregation propensity.

Rugby union referees' physical and physiological demands across different competitive levels.

BACKGROUND: The purpose of this study was to compare the match demands of officiating 15-a-side rugby union at different competitive levels. METHODS: Data was collected using Global Navigation Satellite Systems from 21 referees during 82 competitive rugby union matches across three different competitive levels: 1) professional; 2) semi-professional; 3) amateur. RESULTS: Compared with referees at the professional and semi-professional levels, referees at the amateur level covered less total distance (P=0.005). Additionally, these referees covered less distance jogging, and at low and medium intensity (all P<0.05), and had less time between repeated high-intensity efforts (P<0.001). Furthermore, compared with referees at the semi-professional and amateur levels, referees at the professional level had a higher sprint duration and covered more distance sprinting (all P<0.05), and achieved a higher maximal speed during the longest repeated high-intensity effort or "worst-case scenario" (P=0.026). The professional level referees also displayed a lower average heart rate (P<0.001), spent a higher percentage of time at <60% HRmax (P<0.001), and a lower percentage of time between 71% and 80% HRmax (P=0.004). Finally, external and internal load were strongly correlated for referees at the semi-professional level only (r=0.75, P<0.001). CONCLUSIONS: Overall, the findings demonstrate that refereeing 15-a-side rugby union is more physically demanding at higher competitive levels, particularly in terms of high-intensity efforts. The results provide important information for practitioners involved in the physical preparation and training of rugby union referees.

Laparoscopic Insertion of Various Shaped Trocars in a Porcine Model.

BACKGROUND AND OBJECTIVE: The number of laparoscopic procedures increases annually with an estimated 3% of complications, one third of them linked to Verres' needle or trocar insertion. The safety and efficacy of ports insertion during laparoscopic surgery may be related the technique but also to trocar design. This study aims to compare physical parameters of abdominal wall penetration for 5 different trocars. METHODS: Eleven pigs were studied. Five different commercially available trocars were randomically inserted at the midline. Real-time video recording of the insertions was achieved to measure the excursion of the abdominal wall and the time and distance the cutting surface of the bladed trocars was exposed inside the abdominal cavity. An especially designed hand sensor was developed and placed between the trocar and the hand of the surgeon to record force required for abdominal wall perforation. RESULTS: Greater deformations and forces occurred in nonbladed as compared to bladed trocars, and in conical trocars as compared to pyramidal pointed ones, except for peritoneum perforation. Greater distance and time of blade exposure occurred in pyramidal laminae as compared to conical. CONCLUSION: The bladed trocars have lower forces and deformations in their introduction, and should be those that cause less injury and are more suitable for first entry. Conical and pyramidal trocars with the same blade size showed similar force, deformation, time, and distance of exposed blade.

Discovering Putative Prion-Like Proteins in Plasmodium falciparum: A Computational and Experimental Analysis.

Prions are a singular subset of proteins able to switch between a soluble conformation and a self-perpetuating amyloid state. Traditionally associated with neurodegenerative diseases, increasing evidence indicates that organisms exploit prion-like mechanisms for beneficial purposes. The ability to transit between conformations is encoded in the so-called prion domains, long disordered regions usually enriched in glutamine/asparagine residues. Interestingly, Plasmodium falciparum, the parasite that causes the most virulent form of malaria, is exceptionally rich in proteins bearing long Q/N-rich sequence stretches, accounting for roughly 30% of the proteome. This biased composition suggests that these protein regions might correspond to prion-like domains (PrLDs) and potentially form amyloid assemblies. To investigate this possibility, we performed a stringent computational survey for Q/N-rich PrLDs on P. falciparum. Our data indicate that ∼10% of P. falciparum protein sequences have prionic signatures, and that this subproteome is enriched in regulatory proteins, such as transcription factors and RNA-binding proteins. Furthermore, we experimentally demonstrate for several of the identified PrLDs that, despite their disordered nature, they contain inner short sequences able to spontaneously self-assemble into amyloid-like structures. Although the ability of these sequences to nucleate the conformational conversion of the respective full-length proteins should still be demonstrated, our analysis suggests that, as previously described for other organisms, prion-like proteins might also play a functional role in P. falciparum.

Aerobic power and field test results of amateur 15-a-side rugby union players.

BACKGROUND: The aim of the present study was to verify whether it is possible to predict aerobic power in amateur 15-a-side rugby union players through the Yo-Yo Intermittent Recovery Test Level 1 (Yo-Yo IRT1) and the 5-meter Multiple Shuttle Test (5-m MST). METHODS: Forty-two amateur players - 22 forwards and 20 backs - were evaluated in three phases: 1) maximum treadmill test in the laboratory; 2) field test set by a drawing in the first phase; and 3) second field test. Descriptive, comparison, correlation, regression and level of agreement analyses were performed. RESULTS: Backs, when compared to forwards, showed a higher VO2max (61.7±15 mL/kg/min and 51.6±10.1 mL/kg/min, respectively), Yo-Yo IRT1 final level (16.4±0.8 and 14.9±0.9, respectively) and Yo-Yo IRT1 total distance (1283.3±312.5 m and 792±277.6 m, respectively), and a higher final distance in the 5-m MST (686.8±36.6 and 642.9±46.5, respectively). Significant correlations were found between the result and the total distance on the Yo-Yo IRT1 and the VO2max (r=0.425 and r=0.459, respectively). Using the total distance covered in the Yo-Yo IRT1, the VO2max of amateur 15-a-side rugby union players can be estimated through the equation VO2max = 0.016 × (DIST Yo­Yo) + 40.578. CONCLUSIONS: Yo-Yo IRT1 is most useful when the objective is to evaluate the aerobic power of amateur RU players in comparison with the 5-m MST.

Cavity filling mutations at the thyroxine-binding site dramatically increase transthyretin stability and prevent its aggregation.

More than a hundred different Transthyretin (TTR) mutations are associated with fatal systemic amyloidoses. They destabilize the protein tetrameric structure and promote the extracellular deposition of TTR as pathological amyloid fibrils. So far, only mutations R104H and T119M have been shown to stabilize significantly TTR, acting as disease suppressors. We describe a novel A108V non-pathogenic mutation found in a Portuguese subject. This variant is more stable than wild type TTR both in vitro and in human plasma, a feature that prevents its aggregation. The crystal structure of A108V reveals that this stabilization comes from novel intra and inter subunit contacts involving the thyroxine (T4) binding site. Exploiting this observation, we engineered a A108I mutation that fills the T4 binding cavity, as evidenced in the crystal structure. This synthetic protein becomes one of the most stable TTR variants described so far, with potential application in gene and protein replacement therapies.

Amyloid properties of the leader peptide of variant B cystatin C: implications for Alzheimer and macular degeneration.

Variant B (VB) of cystatin C has a mutation in its signal peptide (A25T), which interferes with its processing leading to reduced secretion and partial retention in the vicinity of the mitochondria. There are genetic evidences of the association of VB with Alzheimer's disease (AD) and age-related macular degeneration (AMD). Here, we investigated aggregation and amyloid propensities of unprocessed VB combining computational and in vitro studies. Aggregation predictors revealed the presence of four aggregation-prone regions, with a strong one at the level of the signal peptide, which indeed formed toxic aggregates and mature amyloid fibrils in solution. In light of these results, we propose for the first time the role of the signal peptide in pathogenesis of AD and AMD.