Extracellular Vesicles as Vehicles of Toxicity in Models of Krabbe Disease
Creators: name: Reiter, Cory Raquel
Publisher: name: University of Illinois at Chicago
Year: 2022
Subjects: Uncategorized

description: Krabbe’s Disease is caused by mutations in the gene encoding the lysosomal enzyme galactosylceramidase, leading to loss of normal catabolism of the prominent myelin lipid galactosylceramide and instead accumulating its intermediate, psychosine (galactosylsphingosine). Toxic accumulation of psychosine is thought to drive pathology in this disease through complex and interwoven mechanisms within the central nervous system. Owing to the physicochemical properties of psychosine, the lipid inserts into membranes causing altered fluidity and architecture with consequential downstream effects. One such outcome is the formation of vesicles at myelin membranes and within the cell, which may be secreted as extracellular vesicles. We hypothesized that brains of the Twitcher mouse model of Krabbe’s Disease would have increased extracellular vesicles which play a role in driving psychosine-mediated pathology. To test this postulate, an inhibitor of vesicle release, GW4869, was given to both wildtype and Twitcher mice and was expected to ameliorate disease. This inhibitor non-competitively blocks neutral sphingomyelinase activity at membranes, thereby depleting ceramide lipid species necessary for vesicle budding. Treated mice were evaluated for disease progression and extracellular vesicles were collected from the brains using newly optimized methods. Twitcher mouse brains contained higher levels of extracellular vesicles, which were significantly decreased following inhibitor treatment. These mice had worsened disease signs, attributable primarily to the earlier onset of an ataxic gait and the pathognomonic tremor. Lipid analyses demonstrated that total ceramides were decreased as intended, while total psychosine levels were unchanged. The proportion of psychosine secreted, however, was significantly reduced in GW4869 treated Twitcher mice, representing the successful decrease in vesicle release. These findings coincide with region specific increases in demyelination, while gliosis remained unchanged. This suggests that extracellular vesicle release mediates psychosine mobility within the CNS, but may serve as an important mechanism utilized by cells to remove toxic lipid for alternative cells to clear. Experiments were begun to assess the cell type-specific contributions of psychosine-rich vesicle release utilizing mixed and enriched glial cell cultures. Mixed glial cultures treated with GW4869 recapitulated decreased vesicle release observed in vivo, but only microglia-enriched cultures had reduced secretion of vesicles in inhibitor treated groups. These preliminary findings suggest a larger role of microglia to psychosine-mediated toxicity in Krabbe’s Disease. Future studies may elucidate the potential of vesicles to transfer psychosine-mediated toxicity and help to delineate the contribution of specific cell types to the vesicle pool, and their susceptibility to psychosine transfer via extracellular vesicles. Utilizing the techniques outlined here, psychosine-enriched vesicles could serve as biomarkers of Krabbe Disease prognosis or progression. Especially with the advent and implementation of gene therapies in treating disease, these tools will be necessary to track mutational correction through maintained lipid homeostasis.