The Effects of Reduced Glial Plasma Membrane Cholesterol on Actin Dynamics: Insights into Neuropathic Mechanisms
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Authors
R. K., Athira
Issue Date
2024
Type
Thesis
Language
en_US
Keywords
Actin Dynamics , Cholesterol Depletion , Neuropathic Models , Schwann Cells
Alternative Title
Abstract
Demyelinating neuropathy, a neurological disorder characterized by progressive loss of myelin, leads to impaired nerve function and sensory deficits. A cure or disease-modifying treatment for patients with demyelinating neuropathy is not available. Hence, investigating the subcellular mechanisms is crucial for therapeutic interventions. Genetic perturbations in Schwann cell proteins are responsible for these disorders. Cholesterol is an essential and rate-limiting lipid for myelin biogenesis, so understanding the interactions between disease-linked glial proteins and cholesterol is a topic of significance. Our previous studies demonstrated that the absence of peripheral myelin protein 22 (PMP22), a neuropathy-causing Schwann cell protein, disrupts cholesterol trafficking and alters the lipid moiety of the Schwann cell plasma membrane (PM). Earlier research have also shown that PMP22 is critical for myelination and cholesterol metabolism. Building upon these findings, our current study explores the intricate relationship between PM cholesterol deficiency and demyelinating neuropathy, specifically emphasizing subcellular actin dynamics. We hypothesize that reduced PM cholesterol alters the actin dynamics and enhances cholesterol uptake proteins and recycling pathways, thereby giving rise to Schwann cell dysfunctions. We used Sciatic nerves and cultured Schwann cells from genotyped wild-type (Wt) and PMP22-deficient mice and normal rats for the current study. To mimic the reduced cholesterol content of the PMP22-deficient glial PM in normal rat Schwann cells, we employed a pharmacological approach. We inhibited de novo cholesterol synthesis by simvastatin treatment (5 µM and 25 µM). By biochemical protein analyses and high-resolution confocal imaging, we detected a significant (p<0.001) increase in the abundance of filamentous actin (F-actin) compared to monomeric actin (G-actin) in cells with depleted PM cholesterol. In addition, statin exposure induced pronounced morphological changes in Schwann cells, leading to branching of the lamellipodia and dense F-actin bundles. The reduced cholesterol levels impair myelination in rat DRG cultures. The biochemical analysis revealed an increase in the expression of essential proteins involved in cholesterol uptake and recycling. Nerve lysates from 3-week-old PMP22-deficient and Trembler J (TrJ), neuropathic model for CMT1E, had elevated levels of F-actin, compared with Wt. By confocal imaging, we detected a pronounced accumulation of F-actin in the tomacula (myelin swellings) in the nerves of PMP22-deficit mice. The findings from the neuropathic samples were substantiated by unbiased proteomic studies from the PMP22-deficient and TrJ neuropathic model, which showed significant alterations in the expression of proteins involved in cholesterol uptake (p<0.05), and perturbations in the pathways associated with actin polymerization and depolymerization. Together, these findings provide novel insights for unraveling the link between the reduced PM cholesterol of neuropathic Schwann cells and subcellular alterations in actin dynamics.