The Cellular Environment Affects Monomeric α-Synuclein Structure.

The presynaptic protein α-synuclein (aSyn) is an 'intrinsically disordered protein' that is highly dynamic in conformation. Transient intramolecular interactions between its charged N and C termini, and between its hydrophobic region and the C terminus, prevent self-association. These interactions inhibit the formation of insoluble inclusions, which are the pathological hallmark of Parkinson's disease and many other synucleinopathies. This review discusses how these intramolecular interactions are influenced by the specific environment aSyn is in. We discuss how charge, pH, calcium, and salt affect the physiological structure of monomeric aSyn, and how they may favour the formation of toxic structures. The more we understand the dynamic conformations of aSyn, the better we can design desperately needed therapeutics to prevent disease progression.

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry Approach to Correlate Local Structure and Aggregation in α-Synuclein.

In Parkinson's disease and other synucleinopathies, α-synuclein misfolds and aggregates. Its intrinsically disordered nature, however, causes it to adopt several meta-stable conformations stabilized by internal hydrogen bonding. Because they interconvert on short timescales, monomeric conformations of disordered proteins are difficult to characterize using common structural techniques. Few techniques can measure the conformations of monomeric α-synuclein, including millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS). Here, we demonstrate a new approach correlating millisecond HDX-MS data with aggregation kinetics to determine the localized structural dynamics that underpin the self-assembly process in full-length wild-type monomeric α-synuclein. Our custom instrumentation and software enabled measurement of the amide hydrogen-exchange rates on the millisecond timescale for wild-type α-synuclein monomer up to residue resolution and under physiological conditions, mimicking those in the extracellular, intracellular, and lysosomal cellular compartments. We applied an empirical correction to normalize measured hydrogen-exchange rates and thus allow comparison between drastically different solution conditions. We characterized the aggregation kinetics and morphology of the resulting fibrils and correlate these with structural changes in the monomer. Applying a correlative approach to connect molecular conformation to aggregation in α-synuclein for the first time, we found that the central C-terminal residues of α-synuclein are driving its nucleation and thus its aggregation. We provide a new approach to link the local structural dynamics of intrinsically disordered proteins to functional attributes, which we evidence with new details on our current understanding of the relationship between the local chemical environment and conformational ensemble bias of monomeric α-synuclein.

Multiple follicular abnormalities in a 1-year old cat consistent with basaloid follicular hamartomas.

BACKGROUND: In humans, basaloid follicular hamartomas are benign follicular tumours, that can be solitary or multiple, in which case they show autosomal dominant inheritance. HYPOTHESIS/OBJECTIVES: This study describes clinical and histopathological findings observed in a young cat, which could be consistent with basaloid follicular hamartomas. CASE DESCRIPTION: Multiple follicular abnormalities, consistent with cutaneous diffuse basaloid follicular hamartomas, were observed in skin samples from a one-year old neutered domestic short hair cat. Clinical signs were diffuse symmetrical alopecia with exaggerated skin markings (ventral abdomen, thorax and medial aspects of the limbs) and intense follicular-centred thickening (face and feet). Microscopic lesions were characterised by multiple proliferative follicular abnormalities in all samples. The epidermis showed a very irregular surface with the follicles filled with variably pigmented keratin. The epithelial walls of the follicles had multiple small hyperplastic basaloid cells foci. In the superficial dermis under the epidermis and around the follicles, fibroblastic spindle-shaped mesenchymal cells with a homogeneous moderate density were present in the collagenous connective tissue. The interfollicular epidermis was also abnormal with multiple small proliferating trichoblastic foci originating from the basal layer. RNAscope testing for feline papillomavirus was negative. CONCLUSIONS AND CLINICAL RELEVANCE: This case report provides the first evidence of clinical and histopathological findings of multiple follicular abnormalities, consistent with cutaneous diffuse basaloid follicular hamartomas in a cat.

De multiples anomalies folliculaires, compatibles avec des hamartomes folliculaires basaloïdes diffus cutanés, ont été observées dans des échantillons de peau d'un chat domestique à poils courts castré âgé d'un an. Les signes cliniques étaient une alopécie diffuse symétrique avec des marques cutanées exagérées (abdomen ventral, thorax et face médiale des membres) et un épaississement folliculaire intense (face et pieds).

Múltiplas anormalidades foliculares, consistentes com hamartomas cutâneos foliculares basaloides difusos, foram observadas em amostras de pele de um gato doméstico de pelo curto castrado de um ano de idade. Os sinais clínicos foram alopecia simétrica difusa com marcações cutâneas exuberantes (abdômen, tórax e aspecto medial dos membros) e espessamento folicular central intenso (face e patas).

Se observaron múltiples anomalías foliculares, consistentes con hamartomas foliculares basaloides difusos cutáneos, en muestras de piel de un gato doméstico de pelo corto castrado de 1 año. Los signos clínicos fueron alopecia simétrica difusa con marcas cutáneas exageradas (abdomen ventral, tórax y cara medial de las extremidades) e intenso engrosamiento de la piel centrado en los folículos (cara y pies).

Different Structural Conformers of Monomeric α-Synuclein Identified after Lyophilizing and Freezing.

Understanding the mechanisms behind amyloid protein aggregation in diseases, such as Parkinson's and Alzheimer's disease, is often hampered by the reproducibility of in vitro assays. Yet, understanding the basic mechanisms of protein misfolding is essential for the development of novel therapeutic strategies. We show here, that for the amyloid protein α-synuclein (aSyn), a protein involved in Parkinson's disease (PD), chromatographic buffers and storage conditions can significantly interfere with the overall structure of the protein and thus affect protein aggregation kinetics. We apply several biophysical and biochemical methods, including size exclusion chromatography (SEC), dynamic light scattering (DLS), and atomic force microscopy (AFM), to characterize the high molecular weight conformers formed during protein purification and storage. We further apply hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to characterize the monomeric form of aSyn and reveal a thus far unknown structural component of aSyn at the C-terminus of the protein. Furthermore, lyophilizing the protein greatly affected the overall structure of this monomeric conformer. We conclude from this study that structural polymorphism may occur under different storage conditions, but knowing the structure of the majority of the protein at the start of each experiment, as well as the factors that may influence it, may pave the way to an improved understanding of the mechanism leading to aSyn pathology in PD.

Anti-NMDA receptor encephalitis presenting as isolated aphasia in an adult.

Anti-NMDA receptor (NMDA-r) encephalitis is a relatively rare cause of autoimmune encephalitis with divergent clinical presentations. We report a case of an adult patient with anti-NMDA-r encephalitis presenting with isolated, abrupt-onset aphasia. Her condition remained unaltered over a period of 6 months. The patients' electroencephalogram findings were typical for NMDA-r encephalitis; however, her magnetic resonance imaging and cerebrospinal fluid analysis were normal. She responded well to immunotherapy, and aphasia eventually resolved. The natural course of the present case contradicts the rapidly progressive nature of typical NMDA-r encephalitis. Furthermore, it broadens the clinical spectrum of anti-NMDA-r encephalitis, to incorporate isolated, nonprogressive aphasia.

Tandem-repeat proteins conformational mechanics are optimized to facilitate functional interactions and complexations.

The architectures of tandem-repeat proteins are distinct from those of globular proteins. Individual modules, each comprising small structural motifs of 20-40 residues, are arrayed in a quasi one-dimensional fashion to form striking, elongated, horseshoe-like, and superhelical architectures, stabilized solely by short-range interaction. The spring-like shapes of repeat arrays point to elastic modes of action, and these proteins function as adapter molecules or 'hubs,' propagating signals within multi-subunit assemblies in diverse biological contexts. This flexibility is apparent in the dramatic variability observed in the structures of tandem-repeat proteins in different complexes. Here, using computational analysis, we demonstrate the striking ability of just one or a few global motions to recapitulate these structures. These findings show how the mechanics of repeat arrays are robustly enabled by their unique architecture. Thus, the repeating architecture has been optimized by evolution to favor functional modes of motions. The global motions enabling functional transitions can be fully visualized at http://bahargroup.org/tr_web.

Influence of point mutations on PR65 conformational adaptability: Insights from molecular simulations and nanoaperture optical tweezers.

PR65 is the HEAT repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem repeat protein. Its conformational mechanics plays a crucial role in PP2A function by opening/closing substrate binding/catalysis interface. Using in silico saturation mutagenesis, we identified PR65 "hinge" residues whose substitutions could alter its conformational adaptability and thereby PP2A function, and selected six mutations that were verified to be expressed and soluble. Molecular simulations and nanoaperture optical tweezers revealed consistent results on the specific effects of the mutations on the structure and dynamics of PR65. Two mutants observed in simulations to stabilize extended/open conformations exhibited higher corner frequencies and lower translational scattering in experiments, indicating a shift toward extended conformations, whereas another displayed the opposite features, confirmed by both simulations and experiments. The study highlights the power of single-molecule nanoaperture-based tweezers integrated with in silico approaches for exploring the effect of mutations on protein structure and dynamics.

Causes of Mortality and Pathological Findings in European Hedgehogs (Erinaceus europaeus) Admitted to a Wildlife Care Centre in Southwestern France from 2019 to 2020.

The European hedgehog (Erinaceus europaeus) is a nocturnal, solitary and non-territorial mammal endemic across Europe and central Asia. Due to population decline in recent decades in Europe, this species was declared as protected and registered as 'Least Concern' on the International Union for Conservation of Nature Red List of Threatened Species. Previous studies pointed to the possible contribution of human activities to hedgehog mortality but few reports provide a complete overview of the pathological processes associated with mortality of the European hedgehog. The aim of this study was to identify the causes of mortality and pathological findings in 35 wild adult European hedgehogs that died spontaneously or were euthanized at the Wildlife Care Centre of the Ecole Nationale Vétérinaire de Toulouse between 2019 and 2020. The main causes of mortality were trauma (41%) including collision (9%), predation (9%), trapping (6%) and trauma of unknown origin (17%). Infectious diseases associated with systemic and local bacterial infections accounted for 34% of the cases and included cutaneous wounds (23%), mandibular abscesses and botryomycosis (23%), exudative bronchopneumonia (9%), suppurative rhinitis (7%) and otitis (3%). One hedgehog (3%) had a large subcutaneous tumour consistent with a malignant peripheral nerve sheath tumour. Parasitism was associated with mortality in 11% of cases with severe debilitation, massive infestation and active larval migration. Incidental findings included splenic and hepatic extramedullary haematopoiesis (100% and 69%, respectively), pulmonary crenosomiasis (91%), gastrointestinal capillariasis (61%), renal lipofuscinosis (59%), chronic renal infarction and interstitial nephritis (50%). These data emphasize the need for further study of hedgehog mortality and of the genetic, behavioural and environmental factors that may contribute to population decline.

Additional Value of Second Harmonic Generation Microscopy in the Diagnosis of Feline Collagen Type III Glomerulopathy.

A 1.5-year-old neutered female Domestic Shorthair cat was euthanized after the diagnosis of end-stage protein-losing nephropathy associated with the onset of nephrotic syndrome. At necropsy, both kidneys were diffusely pale and swollen with a granular cortex. Histologically, glomeruli had diffuse global mesangial and capillary wall expansion by homogeneous pale eosinophilic material. This material was Congo red negative, blue with Masson's trichrome stain, weakly positive with periodic acid-Schiff stain, bright red with Picrosirius red and birefringent under polarized light. Transmission electron microscopy and second harmonic generation (SHG) microscopy revealed mesangial and subendothelial collagen fibril deposition. Type III collagen deposition was confirmed by immunohistochemistry. This study provides an original and complete description of feline collagen type III glomerulopathy and emphasizes the possibility of directly diagnosing glomerular collagen deposition on unstained slides through SHG microscopy.

Extent of N-terminus exposure of monomeric alpha-synuclein determines its aggregation propensity.

As an intrinsically disordered protein, monomeric alpha-synuclein (aSyn) occupies a large conformational space. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. We observe that the more exposed the N-terminus and the beginning of the NAC region of aSyn are, the more aggregation prone monomeric aSyn conformations become. Solvent exposure of the N-terminus of aSyn occurs upon release of C-terminus interactions when calcium binds, but the level of exposure and aSyn's aggregation propensity is sequence and post translational modification dependent. Identifying aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein.

C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction.

Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.

Sequential high gravity ethanol fermentation and anaerobic digestion of steam explosion and organosolv pretreated corn stover.

The present work investigates the suitability of pretreated corn stover (CS) to serve as feedstock for high gravity (HG) ethanol production at solids-content of 24wt%. Steam explosion, with and without the addition of H2SO4, and organosolv pretreated CS samples underwent a liquefaction/saccharification step followed by simultaneous saccharification and fermentation (SSF). Maximum ethanol concentration of ca. 76g/L (78.3% ethanol yield) was obtained from steam exploded CS (SECS) with 0.2% H2SO4. Organosolv pretreated CS (OCS) also resulted in high ethanol concentration of ca. 65g/L (62.3% ethanol yield). Moreover, methane production through anaerobic digestion (AD) was conducted from fermentation residues and resulted in maximum methane yields of ca. 120 and 69mL/g volatile solids (VS) for SECS and OCS samples, respectively. The results indicated that the implementation of a liquefaction/saccharification step before SSF employing a liquefaction reactor seemed to handle HG conditions adequately.