Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all α-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons' disease brain.

Oligonucleotide-based therapeutics have been implemented as a new therapeutic modality in biotech industry, which offers the opportunity to develop formulation platforms for robust parenteral formulations. The aim of this study was to gain a better understanding of stabilizing/de-stabilizing effects of different formulation parameters on unconjugated and N-acetylgalactosamine (GalNAc) conjugated single stranded oligonucleotides with locked nucleic acid modifications (LNA SSO), as model oligonucleotides. Various buffer systems, pH levels and different excipients were evaluated to optimize conditions for LNA SSO in liquid formulations. LNA SSO were exposed to different temperature conditions, mechanical stress as well as oxidative conditions, and the maximum feasible LNA SSO concentrations regarding handling and processing were determined. Finally, options for terminal sterilization of LNA SSO were evaluated. Results show that the tested LNA SSO were most stable under slightly alkaline conditions. A decrease in viscosity was best accomplished in the presence of spermine and lysine. Heat treatment and gamma irradiation caused high levels of degradation of the LNA SSO. Crucial formulation parameters, as identified in this study, should contribute to a significant increase in future productivity in drug product development for single-stranded oligonucleotides.

Summary Parkinson’s disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites, which are neuronal inclusions that are immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of this Lewy pathology is crucial to understanding pathogenesis and progression of the disease. Using correlative light and electron microscopy/tomography on brain tissue from five Parkinson’s disease brain donors, we identified α-synuclein immunopositive Lewy pathology and could show that the majority of these features including Lewy bodies and Lewy neurites primarily consists of a crowded membranous medley of vesicular structures and dysmorphic organelles. Only a small fraction of observed Lewy bodies contained predominant proteinaceous filaments, as previously described. The crowding of organellar components was confirmed by STED- based super-resolution microscopy, and high lipid content within the α-synuclein immunopositive inclusions was corroborated by confocal imaging, CARS/FTIR imaging and lipidomics. Applying this correlative high-resolution imaging and biophysical approach, we discovered in the postmortem brain of Parkinson’s patients a subcellular protein-lipid compartmentalization not previously described in Lewy pathology.

Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines.

Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.

Covalent modifications of therapeutic proteins are of interest for the biotech industry as they potentially impact the quality of the material. This study focuses on covalent protein modifications by the reducing monosaccharide glucose via the glycation reaction. In particular, we examined (i) to which extent different therapeutic monoclonal antibodies are glycated, (ii) the glycation during storage in sucrose-containing formulation buffers where non-reducing sucrose potentially could hydrolyze into its reducing constituent monosaccharides and (iii) the risk of glycation in the course of short-term incubation in Dextrose infusion bags in simulated administration testing. A boronate affinity chromatography method was employed to detect and quantify glycation products in different antibody formulations. For confirmation and to determine the degree of glycation per molecule, selected samples were analyzed via LC-ESI-MS. We could demonstrate that different antibodies differed drastically regarding the degree of glycation, probably a result of their respective fermentation conditions and protein glycation susceptibility. We also demonstrated that sucrose is a non-critical excipient with respect to glycation when stored long-term at intended storage conditions (2-8 degrees C). Finally, we could show that short-term incubation of antibodies in Dextrose infusion bags might lead to huMAb glycation, suggesting to test on glycated products when considering diluting protein drug products in infusion media containing reducing sugars.