Year: 2025
Type: dissertation
Abstract: Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system (CNS) characterized by a disrupted blood-brain barrier (BBB), which is composed of endothelial cells (EC) connected by tight junction (TJ) proteins like claudin 5 and zonula occludens 1 (ZO-1). This structure prevents harmful substances and immune cells from entering the CNS. In both MS and its mouse model, experimental autoimmune encephalomyelitis (EAE), reduced TJ molecule expression increases EC permeability, allowing immune cell infiltration, which produce pro-inflammatory cytokines and cause neuronal damage. Nle4-D-Phe7-α-MSH (NDP-MSH), a synthetic alpha melanocyte-stimulating hormone analogue, ameliorates EAE by binding to melanocortin 1 receptor (MC1R), reducing immune cell infiltration and pro-inflammatory cytokine secretion. Moreover, NDP-MSH was shown to have neuroprotective capacities. However, its effect on BBB integrity remains unclear. This study proposes that enhancing BBB integrity through NDP-MSH reduces immune infiltration, protects neurons, and therefore ameliorates EAE. In accordance with animal welfare law, EAE was induced in C57BL/6 mice and MC1Re/e mice, having a non-functional MC1R, using myelin oligodendrocyte glycoprotein (MOG) peptide. Due to the short half-life of NDP-MSH, the compound was encapsulated into PLGA microparticles (NDP-MSH mp) and its effect was tested in these mice. NDP-MSH mp ameliorated EAE symptoms in an MC1R-dependent manner. Moreover, immunofluorescence staining of brain sections revealed a marked increase in claudin-5 and ZO-1 expression, along with significantly enhanced co-localization in C57BL/6 mice, but not in MC1Re/e mice. In support of this, qPCR experiments confirmed a significant upregulation in Cldn5, Tjp1 mRNA expression and showed significant increase in Ocln and Cdh5 mRNA expression in C57BL/6 mice after NDP-MSH mp treatment, by binding to MC1R, indicating enhanced BBB integrity. Furthermore, trans-endothelial electrical resistance (TEER) assays on murine brain microvascular EC (MBMEC) treated with TNFα and IL-1β demonstrated increased electrical resistance after NDP-MSH mp treatment, suggesting improved EC monolayer integrity. Notably, this effect was abrogated in MBMEC isolated from MC1Re/e mice. Additionally, qPCR analysis revealed elevated Cldn5 and Tjp1 expression in C57BL/6 mice, while MC1Re/e MBMEC showed no significant changes, indicating MC1R-mediated effect. RNA sequencing of brain CD45+ cells from mice with active EAE indicated decreased Vegfa expression, encoding vascular endothelial growth factor A, in C57BL/6 mice, suggesting its involvement in the molecular pathway mediating the effects of NDP-MSH mp. These findings reveal that NDP-MSH mp enhances BBB integrity in EAE via MC1R binding, might provide a potential therapeutic approach for neurodegenerative diseases like MS through PLGA microparticle delivery. Further investigation of the molecular mechanism should be conducted as well as generating PLGA nanoparticle containing NDP-MSH will probably help in improving the therapeutic outcomes.
Author: Alsaedi, Eyad
Language: English
Subfield: Neurology
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7127252554