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.

Elucidating Axonal Injuries Through Molecular Modelling of Myelin Sheaths and Nodes of Ranvier

Around half of the traumatic brain injuries are thought to be axonal damage. Disruption of the cellular membranes, or alternatively cytoskeletal damage has been suggested as possible injury trigger. Here, we have used molecular models to have a better insight on the structural and mechanical properties of axon sub-cellular components. We modelled myelin sheath and node of Ranvier as lipid bilayers at a coarse grained level. We built ex-novo a model for the myelin. Lipid composition and lipid saturation were based on the available experimental data. The model contains 17 different types of lipids, distributed asymmetrically between two leaflets. Molecular dynamics simulations were performed to characterize the myelin and node-of-Ranvier bilayers at equilibrium and under deformation and compared to previous axolemma simulations. We found that the myelin bilayer has a slightly higher area compressibility modulus and higher rupture strain than node of Ranvier. Compared to the axolemma in unmyelinated axon, mechanoporation occurs at 50% higher strain in the myelin and at 23% lower strain in the node of Ranvier in myelinated axon. Combining the results with finite element simulations of the axon, we hypothesizes that myelin does not rupture at the thresholds proposed in the literature for axonal injury while rupture may occur at the node of Ranvier. The findings contribute to increases our knowledge of axonal sub-cellular components and help to understand better the mechanism behind axonal brain injury.

Different Head Group Dependence on The Lipid Thermodynamic Property of Myelin Basic Protein in the Lipid Monolayer

The interaction between the role of 18.5 KDa myelin basic protein (MBP) isoform and phospholipids has been thought to maintain the stability and compactness of the myelin sheath structure. In this study, we describe the statistical thermodynamic theory of different concentrations’ effects on MBP in the major myelin lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)，and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS)) monolayers at the air/subphase interface via Langmuir-Blodgett (LB) technique. A simple statistical mechanical theory is established that predicts the interaction between proteins and phosphate head groups at low surface pressures and the second virial coefficient dependences for the PC, PE, and PS head groups are illustrated. In addition, the surface pressure(π)-mean molecular area(mma) curves were also analyzed using two-dimensional virial equation of state (2D-VES). The positively charged showed that MBP may integrate into different lipid monolayers via hydrophobic and electrostatic interactions, which was found to be consistent with AFM observations of domain and aggregate structures as well as with changes in the surface morphology induced by MBP. These analyses pertaining to membrane structure will provide better insight into membrane modeling systems, especially the interaction between membrane molecules.

Cardiovascular risks impact human brain N-acetylaspartate in regionally specific patterns

Cardiovascular risk factors such as dyslipidemia and hypertension increase the risk for white matter pathology and cognitive decline. We hypothesize that white matter levels of N-acetylaspartate (NAA), a chemical involved in the metabolic pathway for myelin lipid synthesis, could serve as a biomarker that tracks the influence of cardiovascular risk factors on white matter prior to emergence of clinical changes. To test this, we measured levels of NAA across white matter and gray matter in the brain using echo planar spectroscopic imaging (EPSI) in 163 individuals and examined the relationship of regional NAA levels and cardiovascular risk factors as indexed by the Framingham Cardiovascular Risk Score (FCVRS). NAA was strongly and negatively correlated with FCVRS across the brain, but, after accounting for age and sex, the association was found primarily in white matter regions, with additional effects found in the thalamus, hippocampus, and cingulate gyrus. FCVRS was also negatively correlated with creatine levels, again primarily in white matter. The results suggest that cardiovascular risks are related to neurochemistry with a predominantly white matter pattern and some subcortical and cortical gray matter involvement. NAA mapping of the brain may provide early surveillance for the potential subclinical impact of cardiovascular and metabolic risk factors on the brain.

CNS myelination and remyelination depend on fatty acid synthesis by oligodendrocytes

Oligodendrocytes (OLs) support neurons and signal transmission in the central nervous system (CNS) by enwrapping axons with myelin, a lipid-rich membrane structure. We addressed the significance of fatty acid (FA) synthesis in OLs by depleting FA synthase (FASN) from OL progenitor cells (OPCs) in transgenic mice. While we detected no effects in proliferation and differentiation along the postnatal OL lineage, we found that FASN is essential for accurate myelination, including myelin growth. Increasing dietary lipid intake could partially compensate for the FASN deficiency. Furthermore, FASN contributes to correct myelin lipid composition and stability of myelinated axons. Moreover, we depleted FASN specifically in adult OPCs to examine its relevance for remyelination. Applying lysolecithin-induced focal demyelinating spinal cord lesions, we found that FA synthesis is essential to sustain adult OPC-derived OLs and efficient remyelination. We conclude that FA synthesis in OLs plays key roles in CNS myelination and remyelination.

Formalin Tissue Fixation Biases Myelin-Sensitive MRI

PurposeChemical fixatives, such as formalin, form cross-links between proteins and affect the relaxation times and diffusion properties of tissue. These fixation-induced changes likely also affect myelin density measurements produced by quantitative magnetization transfer (qMT) and myelin water imaging (MWI). In this work, we evaluate these myelin-sensitive MRI methods for fixation-induced biases.MethodsWe perform qMT, MWI, and D2O-exchanged zero echo-time (ZTE) imaging on unfixed human spinal cord tissue, and repeat these measurements after 1 day and 31 days of formalin fixation.ResultsThe qMT bound pool fraction increased by 30.7±21.1% after 1 day of fixation and by 42.6±33.9% after 31 days of fixation. Myelin water fraction increased by 39.7±15.5% and 37.0±15.9% at these same time points, and mean T2 of the myelin water pool nearly doubled. Reference-normalized D2O-exchanged ZTE signal intensity increased by 8.17±6.03% after 31 days of fixation, but did not change significantly after 1 day of fixation. After fixation, specimen cross-sectional area decreased by approximately 5%; after correction for shrinkage, changes in D2O-exchanged ZTE intensity were nearly eliminated.ConclusionF and MWF are significantly increased by formalin fixation, while D2O-exchanged ZTE intensity is minimally affected. Changes in qMT and MWI may be due, in part, to delamination and formation of vacuoles in the myelin sheath. D2O-exchanged signal intensity may be altered by fixation-induced changes in myelin lipid solid-state 1H T1. We urge caution in the comparison of these measurements across subjects or specimens in different states, especially unfixed vs. fixed tissue.