Phenotypical and Myopathological Consequences of Compound Heterozygous Missense and Nonsense Variants in SLC18A3

Background: Presynaptic forms of congenital myasthenic syndromes (CMS) due to pathogenic variants in SLC18A3 impairing the synthesis and recycling of acetylcholine (ACh) have recently been described. SLC18A3 encodes the vesicular ACh transporter (VAChT), modulating the active transport of ACh at the neuromuscular junction, and homozygous loss of VAChT leads to lethality. Methods: Exome sequencing (ES) was carried out to identify the molecular genetic cause of the disease in a 5-year-old male patient and histological, immunofluorescence as well as electron- and CARS-microscopic studies were performed to delineate the muscle pathology, which has so far only been studied in VAChT-deficient animal models. Results: ES unraveled compound heterozygous missense and nonsense variants (c.315G>A, p.Trp105* and c.1192G>C, p.Asp398His) in SLC18A3. Comparison with already-published cases suggests a more severe phenotype including impaired motor and cognitive development, possibly related to a more severe effect of the nonsense variant. Therapy with pyridostigmine was only partially effective while 3,4 diaminopyridine showed no effect. Microscopic investigation of the muscle biopsy revealed reduced fibre size and a significant accumulation of lipid droplets. Conclusions: We suggest that nonsense variants have a more detrimental impact on the clinical manifestation of SLC18A3-associated CMS. The impact of pathogenic SLC18A3 variants on muscle fibre integrity beyond the effect of denervation is suggested by the build-up of lipid aggregates. This in turn implicates the importance of proper VAChT-mediated synthesis and recycling of ACh for lipid homeostasis in muscle cells. This hypothesis is further supported by the pathological observations obtained in previously published VAChT-animal models.

BASHY Dye Platform Enables the Fluorescence Bioimaging of Myelin Debris Phagocytosis by Microglia during Demyelination

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the presence of demyelinated regions with accumulated myelin lipid debris. Importantly, to allow effective remyelination, such debris must be cleared by microglia. Therefore, the study of microglial activity with sensitive tools is of great interest to better monitor the MS clinical course. Using a boronic acid-based (BASHY) fluorophore, specific for nonpolar lipid aggregates, we aimed to address BASHY’s ability to label nonpolar myelin debris and image myelin clearance in the context of demyelination. Demyelinated ex vivo organotypic cultures (OCSCs) and primary microglia cells were immunostained to evaluate BASHY’s co-localization with myelin debris and also to evaluate BASHY’s specificity for phagocytosing cells. Additionally, mice induced with experimental autoimmune encephalomyelitis (EAE) were injected with BASHY and posteriorly analyzed to evaluate BASHY+ microglia within demyelinated lesions. Indeed, in our in vitro and ex vivo studies, we showed a significant increase in BASHY labeling in demyelinated OCSCs, mostly co-localized with Iba1-expressing amoeboid/phagocytic microglia. Most importantly, BASHY’s presence was also found within demyelinated areas of EAE mice, essentially co-localizing with lesion-associated Iba1+ cells, evidencing BASHY’s potential for the in vivo bioimaging of myelin clearance and myelin-carrying microglia in regions of active demyelination.

Dehydration Enhances Prebiotic Lipid Remodeling and Vesicle Formation in Acidic Environments

The encapsulation of genetic polymers inside lipid bilayer compartments is a vital step in the emergence of cell-based life. However, even though acidic conditions promote many reactions required for generating prebiotic building blocks, prebiotically-relevant lipids tend to form denser aggregates at acidic pHs rather than prebiotically useful vesicles that exhibit sufficient solute encapsulation. Here we describe how dehydration/rehydration (DR) events, a prebiotically-relevant physicochemical process known to promote polymerization reactions, can remodel dense lipid aggregates into thin-walled vesicles capable of RNA encapsulation even at acidic pHs. Furthermore, DR events appears to favor the encapsulation of RNA within thin-walled vesicles over more lipid-rich vesicles, thus conferring such vesicles a selective advantage.

Histochemical observations in Piper malgassicum (Piperaceae) with a special focus on the epidermis

This is the first contribution about the histochemistry of vegetative and reproductive aerial organs in the genus Piper L. Piper malgassicum accumulates alkaloids and terpenes in the epidermis and the underlying layers of parenchyma, both in the leaves, in the stems and in anthers. Some idioblasts appear to contain a large amount of secondary metabolites. The micro-anatomical analysis showed peculiar secretory structures both in the leaves, in the anthers and in the ovary. Several lipid aggregates, alkaloid droplets and calcium oxalate crystals were observed in leaves and stems, indicating their role in defence strategies, mechanical support, and pollinators attraction. In the anthers, we observed elaioplasts whose content suggest an alternative and indirect function in pollination and defence against micro-organisms. Besides, some lipid aggregates surrounded by microtubules, detected in the anthers, were recognized as lipotubuloids. The tapetum was of secretory type. Alkaloids and terpenes were widely distributed in the plant confirming the important biological role of this type of biomolecules and its functional range. In the anthers, terpene and polyphenol inclusions appeared particularly abundant in the epidermal layer, whereas calcium oxalate crystals were observed close to the ovule in the ovary at maturity.

Lipid type doping of the sponge (L3) mesophase

The polymorphism of lipid aggregates has long attracted detailed study due to the myriad factors that determine the final mesophase observed. This study is driven by the need to understand mesophase behaviour for a number of applications, such as drug delivery and membrane protein crystallography. In the case of the latter, the role of the so-called ‘sponge’ (L3) mesophase has been often noted, but not extensively studied by itself. The L3 mesophase can be formed in monoolein/water systems on the addition of butanediol to water, which partitions the headgroup region of the membrane, and decreases its elastic moduli. Like cubic mesophases, it is bicontinuous, but unlike them, has no long-range translational symmetry. In our present study, we show that the formation of the L3 phase can delicately depend on the addition of dopant lipids to the mesophase. While electrostatically neutral molecules similar in shape to monoolein (DOPE, cholesterol) have little effect on the general mesophase behaviour, others (DOPC, DDM) significantly reduce the region in which it can form. Additionally, we show that by combining cholesterol with the anionic lipid DOPG, it is possible to form the largest stable L3 mesophases observed to date, with correlation lengths over 220 Å.

Magainin 2 and PGLa in Bacterial Membrane Mimics II: Membrane Fusion and Sponge Phase Formation

ABSTRACTWe studied the synergistic mechanism of equimolar mixtures of magainin 2 (MG2a) and PGLa in phosphatidylethanolamine/phosphatidylglycerol mimics of Gram-negative cytoplasmic membranes. In a preceding paper [Pachler et al., Biophys. J. 2019 xxx], we reported on the early onset of parallel heterodimer formation of the two antimicrobial peptides already at low concentrations and the resulting defect formation in membranes. Here, we focus on the structures of the peptide/lipid aggregates occurring in the synergistic regime at elevated peptide concentrations. Using a combination of calorimetric, scattering, electron microscopic and in silico techniques, we demonstrate that the two peptides, even if applied individually, transform originally large unilamellar vesicles into multilamellar vesicles, with a collapsed interbilayer spacing resulting from peptide induced adhesion. Interestingly, the adhesion does not lead to a peptide induced lipid separation of charged and charge neutral species. In addition to this behavior, equimolar mixtures of MG2a and PGLa formed surface-aligned fibril-like structures, which induced adhesion zones between the membranes and the formation of transient fusion stalks in molecular dynamics simulations and a coexisting sponge phase observed by small-angle X-ray scattering. The previously reported increased leakage of lipid vesicles of identical composition in the presence of MG2a/PGLa mixtures is therefore related to a peptide-induced cross-linking of bilayers.STATEMENT OF SIGNIFICANCEWe demonstrate that the synergistic activity of the antimicrobial peptides MG2a and PGLa correlates to the formation of surface-aligned fibril-like peptide aggregates, which cause membrane adhesion, fusion and finally the formation of a sponge phase.

A Comprehensive Study on the Biological Activity of Elderberry Extract and Cyanidin 3-O-Glucoside and Their Interactions with Membranes and Human Serum Albumin

In our research we used the extract from dietary supplement of elderberry (EE) and its dominant anthocyanin—cyanidin 3-O-glucoside (Cy 3-gluc). By interacting with a model membrane that reflects the main lipid composition of tumor membranes, the extract components, including Cy 3-gluc, caused an increase in packing order, mainly in the hydrophilic region of the membrane. It can thus be stated that EE caused a rigidifying effect, which is fundamental for understanding its anticancer and antioxidant activity. This study represents the first attempt to unravel the mechanism of interaction of elderberry extract with membranes. The results of the interaction with human serum albumin (HSA) proved that the studied substance quenches the fluorescence of HSA through a static mechanism in which the main interaction forces are Van der Waals and hydrogen bonding. The antioxidant activity of EE and Cy 3-gluc on liposomal membranes, antiradical properties and ability to inhibited the activity of the enzymes cyclooxygenase-1 and cyclooxygenase-2 were also demonstrated. Moreover, the anticancer activity of EE and Cy 3-gluc on human breast adenocarcinoma cell line were investigated. In addition, EE also exhibited the ability to form lipid aggregates in the form of liposomal capsules that can be applied as carriers of active biological substances, and the highest efficacy of EE encapsulation was obtained for multilayered liposome formulations.