Myelination diseases of the central nervous system: Artificial Axons as in vitro models of chemomechanical cues

Abstract

Myelination is a key biological process wherein glial cells such as oligodendrocytes wrap myelin around neuronal axons, forming an insulative sheath that accelerates signal propagation down the axon. A major obstacle to understanding myelination is the challenge of visualizing and reproducibly quantifying this inherently three-dimensional process in vitro. To this end, Van Vliet et al. previously developed Artificial Axons (AAs), a biocompatible platform consisting of 3D-printed axon mimics that can be ensheathed by oligodendrocytes in vitro. In this thesis, we advance and apply the Artificial Axon platform to create in vitro models of lesion-like environments to elucidate the mechanisms underlying myelination diseases.

First, we improve the existing AA platform to investigate how biophysical cues affect myelin wrapping by rat oligodendrocytes. We build a new high-resolution 3D printer (HR-3DP) that can fabricate AAs with sub-kilopascal elastic moduli and <2 µm diameters. These properties are clinically relevant as prior neuroimaging data from human patients show correlations between demyelinating diseases and changes in brain stiffness, axon diameter, and axon density. An open question is whether these biophysical changes simply act as correlative biomarkers or contribute directly to disease progression. We demonstrate that the extent of myelin ensheathment by rat oligodendrocytes is sensitive to the Young’ modulus, diameter, and density of axons, indicating that each of these biophysical cues may play a causal role in influencing an oligodendrocyte’s propensity to myelinate. We further demonstrate that the responses of oligodendrocytes to pro-myelinating compounds are dependent on axon stiffness, and that the relative ranking of drug efficacies differs between stiff and compliant axons. These results reinforce the importance of studying myelination in mechanically representative environments, and highlight the importance of considering biophysical cues when conducting drug screening studies for pro-myelinating compounds.

Second, we demonstrate the promise of using AAs to model lesion-like environments using human oligodendrocytes. For example, multiple sclerosis (MS) is a demyelinating disease affecting over one million adults in the United States, characterized by the destruction of myelin through a range of immune-mediated mechanisms. The accumulation of myelin debris and inflammatory cytokines in the brains of MS patients is thought to contribute to a growth-inhibitory environment that impairs myelin repair. We used AAs to model the impact of myelin debris and microglia co-culture on myelin ensheathment, recapitulating in vivo results demonstrating a dose-dependent effect of myelin debris on myelin ensheathment. We further demonstrate the compatibility of the AAs with myelination by human oligodendrocytes derived from induced pluripotent stem cells (iPSCs). In particular, we explore the effect of the apolipoprotein (ApoE) genotype on myelin ensheathment, based on clinical data that individuals with the ApoE4 allele exhibit worsened MS prognosis compared to individuals with the ApoE3 allele. Finally, we demonstrate how targeted perturbations to cholesterol metabolism pathways differentially impact ApoE3 vs. ApoE4 human oligodendrocytes. In sum, these results demonstrate the potential of AAs to elucidate the cellular and molecular mechanisms of myelination in the context of human disease.