Amlenetug (Lu AF82422) is a human monoclonal antibody targeting α-synuclein in clinical development for multiple system atrophy. We describe a series of studies that characterize its functional properties and supported its selection as a viable clinical candidate. Amlenetug inhibits seeding induced in mouse primary neurons by various α-synuclein fibrillar assemblies and by aggregates isolated from MSA brain homogenate. In vivo, both co-injection of amlenetug with α-synuclein assemblies in mouse brain and peripheral administration inhibit α-synuclein seeding. Amlenetug inhibits uptake of α-synuclein seeds as well as accumulation of C-terminal truncated α-synuclein seeds and demonstrates binding to monomeric, aggregated, and truncated forms of human α-synuclein. The epitope of amlenetug was mapped to amino acids 112-117 and further characterized by crystallographic structure analysis. Based on our data, we hypothesize that targeting α-synuclein will potentially slow further disease progression by inhibiting further pathology development but be without impact on established pathology and symptoms.

Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a "tropism" for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.

In the brain, α-synuclein (aSN) partitions between free unbound cytosolic and membrane bound forms modulating both its physiological and pathological role and complicating its study due to structural heterogeneity. Here, we use an interdisciplinary, synergistic approach to characterize the properties of aSN:lipid mixtures, isolated aSN:lipid co-structures, and aSN in mammalian cells. Enabled by the isolation of the membrane-bound state, we show that within the previously described N-terminal membrane anchor, membrane interaction relies both on an N-terminal tail (NTT) head group layer insertion of 14 residues and a folded-upon-binding helix at the membrane surface. Both binding events must be present; if, for example, the NTT insertion is lost, the membrane affinity of aSN is severely compromised and formation of aSN:lipid co-structures hampered. In mammalian cells, compromised cooperativity results in lowered membrane association. Thus, avidity within the N-terminal anchor couples N-terminal insertion and helical surface binding, which is crucial for aSN membrane interaction and cellular localization, and may affect membrane fusion.

A hallmark of Parkinson's disease is the presence of Lewy bodies consisting of lipids and proteins, mainly fibrillated α-synuclein (aSN). aSN is an intrinsically disordered protein exerting its physiological role in an ensemble of states, one of which coexists in large assemblies with lipids, recently termed co-structures. Here, we decipher the kinetics of aSN:lipid co-structure formation to decode its mechanism of formation, and we show that the co-structures form with a distinct stoichiometry. Through seeded fibrillation assays, we demonstrate that aSN:lipid co-structures accelerate aSN fibril nucleation compared to lipid vesicles alone. A small-angle X-ray scattering-based model is proposed in which aSN decorates the lipid vesicle surface, yielding properties similar to those of the fibril surface, enhancing fibril nucleation. The delicate balance of aSN structural states close to and on the membrane may under given conditions, e.g., increased local concentrations, be a crucial switching factor between functional and pathological behavior.

Background: Synchrotron radiation (SR) sources provide diverse X-ray methods for the investigation of structure -function relationships in biological macromolecules.Scope of review: Recent developments in SR sources and in the X-ray tools they offer for life sciences are reviewed. Specifically, advances in macromolecular crystallography, small angle X-ray solution scattering, X-ray absorption and fluorescence spectroscopy, and imaging are discussed with examples.Major conclusions: SR sources offer a range of X-ray techniques that can be used in a complementary fashion in studies of biological systems at a wide range of resolutions from atomic to cellular scale. Emerging applications of X-ray techniques include the characterization of disordered proteins, noncrystalline and nonequilibrium systems, elemental imaging of tissues, cells and organs, and detection of time-resolved changes in molecular structures.General significance: X-ray techniques are in the center of hybrid approaches that are used to gain insight into complex problems relating to biomolecular mechanisms, disease and possible therapeutic solutions. This article is part of a Special Issue entitled "Science for Life". Guest Editors: Dr. Austen Angell, Dr. Salvatore Magazti and Dr. Federica Migliardo. (C) 2016 Elsevier B.V. All rights reserved.

In plants heterotrimeric complexes of G proteins (consisting of alpha, beta, and gamma subunits) regulate several signaling pathways including seed germination, seedling development, organ shape and size determination. The alpha subunit has GTP binding and hydrolysis activity and the beta- gamma subunits interact with downstream effectors as a heterodimer. Some structural homology among the plant and mammalian subunits have led to early assumptions about similarities in the activation and transduction mechanisms in the two systems. However, recent evidence on the lack of membrane receptors in plants, the constitutively active state of the plant alpha subunit and the existence of a large family of gamma subunits indicate that the mechanisms involving the plant proteins may be significantly different from those in their mammalian counterparts. In our group the alpha subunit from A. thaliana (AtGPA1), an N-terminal mutant (GPA1t), the gamma subunits (AGG1, AGG2), and the rice gamma subunits (RGG1 and RGG2) were expressed in yeast and bacteria. Absorbance spectroscopy, circular dichroism spectropolarimetry, and dynamic light scattering (DLS) analyses show the structural and stability differences between AtGPA1 and GPA1t as well as among all gamma subunits. DLS, native-PAGE and small angle X-ray scattering measurements reveal the stable oligomeric forms of the proteins in solution indicating possible functional roles for the oligomers. Results also demonstrate the high level of the flexibility in the structures of all subunits. Models for possible roles of different subunits in G protein signaling in plants will be presented. Supported by Turkish Atomic Energy Commission and Instruct, a Landmark ESFRI project.

The heterotrimeric guanine nucleotide-binding proteins (G-proteins) mediate transmission of signals from G protein coupled receptors (GPCR) to effector systems including ion channels, enzymes and intracellular second messengers in yeast, mammals and plants. The complex is comprised of alpha (Gα), beta (Gβ) and gamma (Gγ) subunits; Gα has GTP binding and hydrolysis activity, and Gβ and Gγ interact with downstream effectors as a dimeric complex. Although the structure and activation mechanism for the mammalian complex are well known, these are still not fully understood in plants. We investigate biochemical and structural features of heterotrimeric G –proteins from A.thaliana to gain insight into its activation mechanism and to develop a better understanding of molecular interactions in the G-protein related signaling pathways in plants1. Here we present the results of biochemical and structural characterization studies on the wild type AtGPA1 and an N-terminal 36-aa residue truncated version, GPA1t, produced in yeast and bacteria expression systems, respectively. Nucleotide (GTP, GDP, GTPγS) binding is confirmed by absorbance spectroscopy and Circular Dichroism Spectropolarimetry (CD) results indicate that the secondary structure of AtGPA1-GTPγS is more stable than that of AtGPA1-GDP. Dynamic light scattering (DLS) and native-Page analyses combined with small angle X-ray scattering (SAXS) measurements reveal that AtGPA1 has a tendency to form trimers in solution. SAXS data also shows that AtGPA1-GDP has a globular structure with some flexibility. The physiological significance of the trimeric structure and models calculated using SAXS data will be presented. Results will be discussed together with the crystal structure of truncated GPA12, on which the current proposed models of G-protein activation mechanism in plants are based. 1 B. Kaplan (2009), PhD Thesis Sabanci University. 2 J. C. Jones et al.(2011) Sci. Signal., vol. 4, p. ra8.