MD2-TLR4-IN-1 mitigates synuclein preformed fibril-mediated inflammatory response in microglia: insights from an in vitro and in vivo study

Neuroinflammation is a key pathological feature of neurodegenerative diseases, including Parkinson’s disease (PD), where α-synuclein activates microglia via TLRs, leading to inflammatory cytokine release and neuronal damage. In this study, we evaluated the therapeutic potential of MD2-TLR4-IN-1 against α-Syn PFF-induced neuroinflammation in N9 microglial cells and a mouse model. The anti-neuroinflammatory and therapeutic effects of MD2-TLR4-IN-1 were evaluated using in vitro and in vivo models. In vitro studies in N9 microglial and SH-SY5Y neuronal cells included cytotoxicity assessment, Western blotting, and ELISA to analyze pro-inflammatory cytokines and microglial markers. In vivo, α-synuclein PFF-induced mice were assessed for locomotor and motor functions, along with biochemical and immunofluorescence analyses to evaluate tyrosine hydroxylase, α-synuclein, and TLR4 signaling proteins. In vitro studies demonstrated that MD2-TLR4-IN-1 was non-cytotoxic in neuronal cells and significantly modulates M1and M2 microglia -associated markers. In vivo, treatment did not produce statistically significant changes in motor and locomotor activity but significantly downregulated α-synuclein PFF-induced activation of the TLR4 signaling pathway. Additionally, TH expression was nonsignificant in the treatment groups compared with the disease group. MD2-TLR4-IN-1 effectively modulate TLR4 signaling in α-synuclein PFF-induced neuroinflammation. Both in vitro and in vivo findings demonstrate its anti-neuroinflammatory activity and its ability to modulate α-synuclein-associated inflammatory pathology, highlighting its potential as a candidate anti-neuroinflammatory agent for PD. α-Synuclein pre-formed fibrils induce TLR4-associated neuroinflammatory signalingMD2-TLR4-IN-1 target the TLR4/MYD88/NF-kB pathway and modulates inflammatory signalingMD2-TLR4-IN-1 reduces pro-inflammatory mediators and modulates microglial-associated markersMolecular findings support TLR4 pathway modulation, while functional and neuroprotective effects require further validation α-Synuclein pre-formed fibrils induce TLR4-associated neuroinflammatory signaling MD2-TLR4-IN-1 target the TLR4/MYD88/NF-kB pathway and modulates inflammatory signaling MD2-TLR4-IN-1 reduces pro-inflammatory mediators and modulates microglial-associated markers Molecular findings support TLR4 pathway modulation, while functional and neuroprotective effects require further validation